Antibodies specific to human poliovirus receptor (pvr)

ABSTRACT

The present invention provides monoclonal antibodies that recognize poliovirus receptor (PVR) and inhibit its binding to T cell immunoreceptor with Ig and ITIM domains (TIGIT). The present invention further provides pharmaceutical compositions comprising the antibodies and methods for their use in cancer immunotherapy, treating infections and in diagnosis.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a Continuation of U.S. application Ser. No.17/118,062 filed on Dec. 10, 2020, which is a Divisional of U.S.application Ser. No. 16/081,667 filed on Aug. 31, 2018, now U.S. Pat.No. 10,906,987, which is a 35 U.S.C. 371 National Phase EntryApplication from PCT/IL2017/050256, filed Feb. 28, 2017, which claimspriority to U.S. Provisional Application No. 62/364,924, filed Jul. 21,2016 and U.S. Provisional Application No. 62/301,727, filed Mar. 1,2016. The entirety of the disclosure of the above-referencedapplications are incorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted via Patent Center and is hereby incorporated by reference inits entirety. Said Sequence Listing, created on Aug. 13, 2023, is namedYISSUM0137 PCT.xml and is 91,542 bytes in size.

FIELD OF THE INVENTION

The invention is in the field of immunotherapy and relates to antibodiesand fragments thereof which bind to the protein poliovirus receptor, topolynucleotide sequences encoding these antibodies and to hybridomacells producing these antibodies. The invention further relates totherapeutic and diagnostic compositions comprising these antibodies andto methods of treating and diagnosing diseases, particularly cancer,using these monoclonal antibodies.

BACKGROUND OF THE INVENTION

Cancer immunotherapy is utilized for generating and augmenting ananti-tumor immune response, e.g., by treatment with antibodies specificto antigens on tumor cells, with fusions of antigen presenting cellswith tumor cells, or by specific activation of anti-tumor T cells. Theability of recruiting immune cells (e.g. T cells) against tumor cells ina patient provides a therapeutic modality of fighting cancer types andmetastasis that so far were considered incurable.

T cell mediated immune response includes multiple sequential stepsregulated by a balance between co-stimulatory and co-inhibitory signalsthat control the magnitude of the immune response. The inhibitorysignals, referred to as immune checkpoints, are crucial for themaintenance of self-tolerance and also for the limitation ofimmune-mediated collateral tissue damage. These signals change as aninfection or immune provocation is cleared, worsens, or persists, andthese changes affect the response of T cells and re-shape the immuneresponse.

The expression of immune checkpoint proteins can be regulated by tumors.For example, upregulation of programmed death-1 ligand (PD-L1) on thecancer cell surface allows them to evade the host immune system byinhibiting T cells via binding to PD-1 that might otherwise attack thesetumor cells. Thus, immune checkpoints represent significant barriers toactivation of functional cellular immunity against cancer. Accordingly,antagonistic antibodies specific for inhibitory ligands on immune cellsare considered viable anti-cancer agents and they are being evaluated inthe clinics (e.g. Nivolumab and Pembrolizumab). Another example forimmune checkpoint molecule is T cell immunoreceptor with Ig and ITIMdomains (TIGIT). TIGIT is a co-inhibitory molecule expressed on variousimmune cells including T cells and Natural Killer cells (NK cells).TIGIT binds with high affinity to polio virus receptor (PVR).

Poliovirus receptor (PVR), also termed CD155, is a transmembraneglycoprotein involved in mediating cell adhesion to extracellular matrixmolecules. It was previously described as a tumor antigen and as apotential target for therapeutic intervention as its expression isup-regulated in neuroectodermal cancers, including glioblastomamultiforme, medulloblastoma, and colorectal carcinoma (Solecki et al.,J. Biol. Chem. 2002, 277: 25697-700) as well as in pancreatic cancer(Nishiwada et al., Anticancer Res. 2015, 35(4): 2287-97). PVR is alsoknown to enhance the serum-induced activation of the Ras-Raf-MEK-ERKsignaling, up-regulating cyclins D2 and E, and down-regulated p27Kip1,eventually shortening the period of the G0/G1 phase of the cell cycle(Kakunaga 2004, J. Biological Chemistry, 279, 36419-36425) for thatreason blocking of PVR on tumor cells is anticipated to reduce viabilityof tumor cells. PVR has also a critical role in angiogenesis and issuggested to regulate the VEGF-induced angiogenesis by controlling theinteraction of VEGFR2 with integrin α(v)β(3), and the VEGFR2-mediatedRapi-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012,110(5), 716-26). Additionally, PVR is complexing with IGF1R andparticipating in Met signaling and blocking the complex formationreduced cell viability and angiogenesis (Lee et al., Scientific Reports2014, 20, 4, 7139).

PVR involvement in metastasis was demonstrated by injecting cancer cellsto the tail of mice and measuring metastasis to the lungs. It has beenshown that the upregulated PVR in cancer cells transinteracts with itscounter-receptor in platelets, and that this trans-interaction enhancesthe metastasis of the cancer cells to the lungs (Morimoto et al.,Oncogene (2008) 27, 264-273).

U.S. Patent Application No. 20070041985 discloses molecules specificallybinding to at least one intra- or extracellular domain of the PVR or anyderivative thereof, wherein the molecule has the ability to modulate areceptor mediated adhesion, trafficking and/or invasion behavior of acell expressing the PVR or any derivative thereof.

U.S. Patent Application No. 20090215175 provides molecules (e.g. smallchemical compounds, oligonucleotides, polypeptides, antibodies, andantibody fragments) which modulate the PVR functions necessary foradhesion, trafficking, invasion and/or metastatic potential of cells.The molecules can be used for the treatment of cells having a metastaticpotential, metastasis and cancer.

PCT Application Publication No. WO 2006/124667 discloses modulation ofthe protein zB7R1 (TIGIT) by monoclonal antibodies that block TIGITbinding to its ligand PVR.

There is an unmet need to provide additional and more effective,specific, safe and/or stable agents that alone or in combination withother agents, potentiate cells of the immune system to attack tumor orvirus infected cells by inhibiting PVR binding to TIGIT.

SUMMARY OF THE INVENTION

The present invention provides antibodies and fragments thereof thatrecognize the poliovirus receptor (PVR), prevent its binding to T cellimmunoreceptor with Ig and ITIM domains (TIGIT) and inhibit suppressiveactivity on lymphocytes such as natural killer (NK) cells and T-cells.The anti-PVR antibodies disclosed herein are capable of binding to PVRpresent on cancer cells. These antibodies and fragment thereof arecharacterized by having unique sets of complementarity-determiningregions (CDR) sequences, high affinity and high specificity to PVR andare useful in cancer immunotherapy for combating tumor immune evasion,as stand-alone therapy and in combination with other anti-cancer agents.The antibodies are also useful in treating viral infections.

It is now disclosed that the high affinity anti-PVR antibodies disclosedherein block TIGIT-PVR interaction and restore T and NK cells activity.The antibodies showed high specificity to human PVR. These propertiesmake the monoclonal antibodies (mAbs) of the present invention valuablecandidates for use in cancer immune-therapy, enabling administration oflower doses with fewer side effects.

Advantageously, the anti-PVR mAbs according to the invention can induceT cells proliferation better than anti PD-1 and CTLA-4 mAbs in a PD-L1in-vitro model (A549). The induction effect was shown for peripheralmononuclear blood cells (PMBCs) and purified CD4 and CD8 T cells. Inaddition, PVR mAbs were able to induce NK cell activation in most targetcells tested. Moreover, some of the anti-PVR antibodies described hereinhave comparable anti-cancer activity in-vitro to those of a known agent,Erbitux® currently used in therapy. Furthermore, some of the anti-PVRantibodies described herein showed synergistic effect when combined withadditional anti-cancer agents, such as anti PD-1 and CTLA-1 antibodiesand epidermal growth factor receptor (EGFR). In addition, some of theanti-PVR antibodies were found to induce antibody-dependentcell-mediated cytotoxicity (ADCC). It is further disclosed that someanti-PVR antibodies according to the invention had no blocking effect onthe co-stimulatory signaling of DNAM1, therefore they have nodeleterious effect on other immune induction signals.

Interestingly, despite high sequence similarity between human and rodentPVR sequences, the antibodies of the present invention are highlyspecific to human PVR.

It is further disclosed that unexpectedly some chimeric monoclonalantibodies, comprising human constant chain, showed enhanced effect onimmune cell activation in comparison to their equivalent murinemonoclonal antibodies.

Some of the anti PVR mAbs described herein were able to reduce tumorcells viability in an immune independent manner by blocking of PVR ontumor cells. Without wishing to be bound to any mechanism of action, itis suggested that this activity results from the ability of PVR toshortening the period of the G0/G1 phase of the cell cycle.

According to one aspect, the present invention provides an isolatedmonoclonal antibody which binds to poliovirus receptor (PVR), or anantibody fragment thereof comprising at least the antigen bindingportion, wherein the isolated antibody or antibody fragment is selectedfrom the group consisting of:

-   -   i. three CDRs of a heavy-chain (HC) variable region comprising        SEQ ID NO: 77 and three CDRs of a light-chain (LC) variable        comprising SEQ ID NO: 79, or an analog or derivative thereof        having at least 90% sequence identity with said antibody or        fragment sequence;    -   ii. three complementarity determining regions (CDRs) of a        heavy-chain variable region comprising SEQ ID NO: 69 and three        CDRs of a light-chain variable region comprising SEQ ID NO: 71,        or an analog or derivative thereof having at least 90% sequence        identity with said antibody or fragment sequence; and    -   iii. three CDRs of a heavy-chain variable region comprising SEQ        ID NO: 73 and three CDRs of a light-chain variable region        comprising SEQ ID NO: 75, or an analog or derivative thereof        having at least 90% sequence identity with said antibody or        fragment sequence.

Antibodies comprising CDR sequences contained in heavy and light chainshomologues to SEQ ID Nos: 2, 10, 18, 26, 34 or 42 are also includedwithin the scope of the present invention. According to someembodiments, SEQ ID NO: 2 is interchangeable with SEQ ID NO: 69.According to some embodiments, SEQ ID NO: 10 is interchangeable with SEQID NO: 71. According to some embodiments, SEQ ID NO: 18 isinterchangeable with SEQ ID NO: 73. According to some embodiments, SEQID NO: 26 is interchangeable with SEQ ID NO: 75. According to someembodiments, SEQ ID NO: 34 is interchangeable with SEQ ID NO: 77.According to some embodiments, SEQ ID NO: 42 is interchangeable with SEQID NO: 79.

There are several methods known in the art for determining the CDRsequences of a given antibody molecule, but there is no standardunequivocal method. Determination of CDR sequences from antibody heavyand light chain variable regions can be made according to any methodknown in the art, including but not limited to the methods known asKABAT, Chothia and IMGT. A selected set of CDRs may include sequencesidentified by more than one method, namely, some CDR sequences may bedetermined using KABAT and some using IMGT, for example.

According to some embodiments, the isolated monoclonal antibody orfragment comprises the CDR sequences of a monoclonal antibody denotedAnti-PVR 4E5 (or hPVR.07), namely, the three CDR sequences contained inheavy chain variable region set forth in SEQ ID NO: 69 and the three CDRsequences contained in light chain variable region set forth in SEQ IDNO: 71.

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises heavy-chain CDR1 comprising the sequenceGFDFSRYW (SEQ ID NO: 4). According to some embodiments, the isolatedmonoclonal antibody or the antibody fragment comprises heavy-chain CDR2comprising the sequence EIHPDSSKINYTPSQ (SEQ ID NO: 6). According tosome embodiments, the isolated monoclonal antibody or the antibodyfragment comprises heavy-chain CDR3 comprising the sequence PDGNYNALDYW(SEQ ID NO: 8).

According to some embodiments, the isolated monoclonal antibody orantibody fragment comprises heavy-chain CDR1 comprising the sequenceRYW. According to some embodiments, the isolated monoclonal antibody orthe antibody fragment comprises heavy-chain CDR1 comprising the sequenceRYWMT (SEQ ID NO: 80). According to some embodiments, the isolatedmonoclonal antibody or the antibody fragment comprises heavy-chain CDR3comprising the sequence PDGNYNALDY (SEQ ID NO: 82).

According to certain embodiments, the isolated monoclonal antibody orthe antibody fragment comprises: (i) HC CDR1 comprising the sequenceGFDFSRYW (SEQ ID NO: 4); (ii) HC CDR2 comprising the sequence:EIHPDSSKINYTPSQ (SEQ ID NO: 6); and (iii) HC CDR3 comprising thesequence: PDGNYNALDYW (SEQ ID NO: 8).

According to certain embodiments, the isolated monoclonal antibody orthe antibody fragment comprises: (i) HC CDR1 comprising the sequenceRYW; (ii) HC CDR2 comprising the sequence: EIHPDSSKINYTPSQ (SEQ ID NO:6); and (iii) HC CDR3 comprising the sequence: PDGNYNALDY (SEQ ID NO:82).

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises light-chain CDR1 comprising the sequenceKASQDVGTAVT (SEQ ID NO: 12). According to some embodiments, the isolatedmonoclonal antibody or the antibody fragment comprises light-chain CDR2comprising the sequence WASTRHT (SEQ ID NO: 14). According to someembodiments, the isolated monoclonal antibody or the antibody fragmentcomprises light-chain CDR3 comprising the sequence QQYSRYPYT (SEQ ID NO:16). According to certain embodiments, the isolated monoclonal antibodyor the antibody fragment comprises: (i) LC CDR1 comprises the sequenceKASQDVGTAVT (SEQ ID NO: 12); (ii) LC CDR2 comprises the sequence:WASTRHT (SEQ ID NO: 14); and (iii) HC CDR3 comprises the sequence:QQYSRYPYT (SEQ ID NO: 16).

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises heavy chain CDR1 sequence comprising the sequence:GFDFSRYW (SEQ ID NO: 4), heavy chain CDR2 comprising the sequence:EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 comprising thesequence: PDGNYNALDYW (SEQ ID NO: 8), light chain CDR1 comprising thesequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 comprising thesequence: WASTRHT (SEQ ID NO: 14), and light chain CDR3 comprising thesequence: QQYSRYPYT (SEQ ID NO: 16), or analogs thereof comprising nomore than 5% amino acid substitution, deletion and/or insertion in thehypervariable region (HVR) sequence.

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises a set of six CDR sequences consisting of:

-   -   i. heavy chain CDR1 having a sequence selected from the group        consisting of SEQ ID NO: 4 and SEQ ID NO: 80,    -   ii. heavy chain CDR2 having a sequence selected from the group        consisting of SEQ ID NO: 6 and SEQ ID NO: 81,    -   iii. heavy chain CDR3 having a sequence selected from the group        consisting of SEQ ID NO: 8 and SEQ ID NO: 82,    -   iv. light chain CDR1 having a sequence set forth in SEQ ID NO:        12,    -   v. light chain CDR2 having a sequence set forth in SEQ ID NO:        14, and    -   vi. light chain CDR3 having a sequence set forth in SEQ ID NO:        16.

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises a set of six CDR sequences consisting of: SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, and SEQID NO: 16.

According to other specific embodiments the isolated monoclonal antibodyor fragment comprises a set of six CDR sequences consisting of: SEQ IDNO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 12, SEQ ID NO: 14, andSEQ ID NO: 16.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises heavy chain variable region set forth in SEQID NO: 69), or an analog or derivative thereof having at least 90%sequence identity with the heavy chain variable region sequence.

According to some embodiments, the analog of SEQ ID NO: 2 is heavy chainvariable region having a sequence set forth in SEQ ID NO: 69.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises light chain variable region set forth in SEQID NO: 71), or an analog thereof having at least 90% sequence identitywith the light chain variable region sequence.

According to some embodiments, the analog of SEQ ID NO: 10 is lightchain variable region having a sequence set forth in SEQ ID NO: 71.

According to a specific embodiment, the isolated monoclonal antibody orfragment thereof comprises a heavy chain variable region having asequence set forth in SEQ ID NO: 2 or SEQ ID NO: 69, and a light chainvariable region having a sequence set forth in SEQ ID NO: 10 or SEQ IDNO: 71, or an analog thereof having at least 90% sequence identity withthe light and/or heavy chain sequence.

The invention also encompasses antibody or antibody fragment capable ofbinding with high affinity to an epitope within the human PVR protein towhich monoclonal antibody (mAb) 4E5 binds.

According to other embodiments, the isolated monoclonal antibodycomprises the CDR sequences of a monoclonal antibody denoted 7D4 (orhPVR.01), namely, the three CDR sequences contained in heavy chainvariable region set forth in SEQ ID NO: 73 and the three CDR sequencescontained in light chain variable region set forth in SEQ ID NO: 75.

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises heavy-chain CDR1 comprising the sequenceGYTFTEYTMH (SEQ ID NO: 20). According to some embodiments, the isolatedmonoclonal antibody or the antibody fragment comprises heavy-chain CDR2comprising the sequence GIDPNNGGTNYNQNFKG (SEQ ID NO: 22). According tosome embodiments, the isolated monoclonal antibody or the antibodyfragment comprises heavy-chain CDR3 comprising the sequence VIPLEY (SEQID NO: 24). According to certain embodiments, the isolated monoclonalantibody or the antibody fragment comprises: (i) HC CDR1 comprises thesequence GYTFTEYTMH (SEQ ID NO: 20); (ii) HC CDR2 comprises thesequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); and (iii) HC CDR3 comprisesthe sequence: VIPLEY (SEQ ID NO: 24).

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises heavy-chain CDR1 comprising the sequenceEYTMH (SEQ ID NO: 83).

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises light-chain CDR1 comprising the sequenceKASQNVYTNVA (SEQ ID NO: 28). According to some embodiments, the isolatedmonoclonal antibody or the antibody fragment comprises light-chain CDR2comprising the sequence SASYRYR (SEQ ID NO: 30). According to someembodiments, the isolated monoclonal antibody or the antibody fragmentcomprises light-chain CDR3 comprising the sequence QQYNSYPLA (SEQ ID NO:32). According to certain embodiments, the isolated monoclonal antibodyor the antibody fragment comprises: (i) LC CDR1 comprises the sequenceKASQNVYTNVA (SEQ ID NO: 28); (ii) LC CDR2 comprises the sequence:SASYRYR (SEQ ID NO: 30); and (iii) HC CDR3 comprises the sequence:QQYNSYPLA (SEQ ID NO: 32).

According to some specific embodiments the isolated monoclonal antibodycomprises heavy chain CDR1 comprising the sequence GYTFTEYTMH (SEQ IDNO: 20), heavy chain CDR2 comprising the sequence: GIDPNNGGTNYNQNFKG(SEQ ID NO: 22), heavy chain CDR3 comprising the sequence: VIPLEY (SEQID NO: 24), light chain CDR1 comprising the sequence: KASQNVYTNVA (SEQID NO: 28), light chain CDR2 comprising the sequence: SASYRYR (SEQ IDNO: 30), and light chain CDR3 comprising the sequence: QQYNSYPLA (SEQ IDNO: 32), or analogs thereof comprising no more than 5% amino acidsubstitution, deletion and/or insertion in the HVR sequence.

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises a set of six CDR sequences consisting of: heavychain CDR1 having a sequence selected from the group consisting of SEQID NO: 20 and SEQ ID NO: 83, heavy chain CDR2 having a sequence setforth in SEQ ID NO: 22, heavy chain CDR3 having a sequence set forth inSEQ ID NO: 24, light chain CDR1 having a sequence set forth in SEQ IDNO: 28, light chain CDR2 having a sequence set forth in SEQ ID NO: 30,and light chain CDR3 having a sequence set forth in SEQ ID NO: 32.

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises a set of six CDR sequences consisting of: SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, andSEQ ID NO: 32.

According to other specific embodiments the isolated monoclonal antibodyor fragment comprises a set of six CDR sequences consisting of: SEQ IDNO: 83, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, andSEQ ID NO: 32.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises heavy chain variable region set forth in SEQID NO: 73, or an analog or derivative thereof having at least 90%sequence identity with the heavy chain variable region sequence.

According to some embodiments, the analog of SEQ ID NO: 18 is heavychain variable region having a sequence set forth in SEQ ID NO: 73.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises light chain variable region set forth in SEQID NO: 75, or an analog thereof having at least 90% sequence identitywith the light chain variable region sequence.

According to some embodiments, the analog of SEQ ID NO: 26 is lightchain variable region having a sequence set forth in SEQ ID NO: 75

According to a specific embodiment, the isolated monoclonal antibody orfragment thereof comprises a heavy chain variable region having asequence set forth in SEQ ID NO: 18 or SEQ ID NO: 73, and a light chainvariable region having a sequence set forth in SEQ ID NO: 26 or SEQ IDNO: 75, or an analog thereof having at least 90% sequence identity withthe light and/or heavy chain sequence.

The invention also encompasses antibody or antibody fragment capable ofbinding with high affinity to an epitope within the human PVR protein towhich mAb 7D4 binds.

According to other embodiments, the isolated monoclonal antibodycomprises the CDR sequences of a monoclonal antibody denoted 5B9 (orhPVR.09), namely, the three CDR sequences contained in heavy chainvariable region set forth in SEQ ID NO: 77 and the three CDR sequencescontained in light chain variable region set forth in SEQ ID NO: 79.

According to some embodiments, the isolated monoclonal antibodycomprises the complementarity determining region (CDR) sequencescontained in heavy chain variable region set forth in SEQ ID NO: 34 andthe three CDR sequences contained in light chain variable regionsequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQID NO: 54. Each possibility represents a separate embodiment of theinvention.

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises heavy-chain CDR1 comprising the sequenceGYTFSNYWIE (SEQ ID NO: 36). According to some embodiments, the isolatedmonoclonal antibody or the antibody fragment comprises heavy-chain CDR2comprising the sequence EIFPGSGRINFNEKFKG (SEQ ID NO: 38). According tosome embodiments, the isolated monoclonal antibody or the antibodyfragment comprises heavy-chain CDR3 comprising the sequence TKIYGNSFDY(SEQ ID NO: 40). According to some embodiments, the isolated monoclonalantibody or the antibody fragment comprises heavy-chain CDR1 comprisingthe sequence SNYWIE (SEQ ID NO: 84).

According to certain embodiments, the isolated monoclonal antibody orthe antibody fragment comprises: (i) HC CDR1 comprises the sequenceSNYWIE (SEQ ID NO: 84); (ii) HC CDR2 comprises the sequence:EIFPGSGRINFNEKFKG (SEQ ID NO: 38); and (iii) HC CDR3 comprises thesequence: TKIYGNSFDY (SEQ ID NO: 40).

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises light-chain CDR1 comprising the sequenceKASQDVGTAVV (SEQ ID NO: 44). According to some embodiments, the isolatedmonoclonal antibody or the antibody fragment comprises light-chain CDR2comprising the sequence WASSRHN (SEQ ID NO: 46). According to someembodiments, the isolated monoclonal antibody or the antibody fragmentcomprises light-chain CDR3 comprising the sequence QQYSRYPLT (SEQ ID NO:48).

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises light-chain CDR1 comprising the sequenceKASQDVGTAV (SEQ ID NO: 85).

According to certain embodiments, the isolated monoclonal antibody orthe antibody fragment comprises: (i) LC CDR1 comprises the sequenceKASQDVGTAV (SEQ ID NO: 85); (ii) LC CDR2 comprises the sequence: WASSRHN(SEQ ID NO: 46); and (iii) HC CDR3 comprises the sequence: QQYSRYPLT(SEQ ID NO: 48).

According to additional embodiments, LC CDR2 comprises the sequence setforth in SEQ ID Nos: 46, 56, 57, 58, 59, 60, or 61. Each possibilityrepresents a separate embodiment of the invention.

According to some specific embodiments the isolated monoclonal antibodyor fragment comprises heavy chain CDR1 sequence comprising the sequence:GYTFSNYWIE (SEQ ID NO: 36), heavy chain CDR2 comprising the sequence:EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 comprising thesequence: TKIYGNSFDY (SEQ ID NO: 40), light chain CDR1 comprising thesequence: KASQDVGTAVV (SEQ ID NO: 44), light chain CDR2 comprising thesequence: WASSRHN (SEQ ID NO: 46), and light chain CDR3 comprising thesequence: QQYSRYPLT (SEQ ID NO: 48), or analogs thereof comprising nomore than 5% amino acid substitution, deletion and/or insertion in theHVR sequence.

According to some specific embodiments the isolated monoclonal antibodyor fragment consisting of: heavy chain CDR1 having a sequence selectedfrom the group consisting of SEQ ID NO: 36 and SEQ ID NO: 84, heavychain CDR2 having a sequence set forth in SEQ ID NO: 38, heavy chainCDR3 having a sequence set forth in SEQ ID NO: 40, light chain CDR1having a sequence selected from the group consisting of SEQ ID NO: 44and SEQ ID NO: 85, light chain CDR2 having a sequence set forth in SEQID NO: 46, and light chain CDR3 having a sequence set forth in SEQ IDNO: 48.

According to some specific embodiments the isolated monoclonal antibodyor fragment consisting of: heavy chain CDR1 having a sequence set forthin SEQ ID NO: 36 or SEQ ID NO: 84, heavy chain CDR2 having a sequenceset forth in SEQ ID NO: 38, heavy chain CDR3 having a sequence set forthin SEQ ID NO: 40, light chain CDR1 having a sequence set forth in SEQ IDNO: 44 or SEQ ID NO: 85, light chain CDR2 having a sequence selectedfrom the group consisting of: SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61; andlight chain CDR3 having a sequence set forth in SEQ ID NO: 48. Eachpossibility represents a separate embodiment of the invention.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises heavy chain variable region set forth in SEQID NO: 77, or an analog or derivative thereof having at least 90%sequence identity with the heavy chain variable region sequenceAccording to some embodiments, the analog of SEQ ID NO: 34 is heavychain variable region having a sequence set forth in SEQ ID NO: 77.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises light chain variable region set forth in SEQID NO: 79, or an analog thereof having at least 90% sequence identitywith the light chain variable region sequence.

According to some embodiments, the analog of SEQ ID NO: 42 is lightchain variable region having a sequence set forth in SEQ ID NO: 79.

According to a specific embodiment, the isolated monoclonal antibody orfragment thereof comprises a heavy chain variable region having asequence selected from the group consisting of SEQ ID NO: 34 and SEQ IDNO: 77, and a light chain variable region having a sequence selectedfrom the group consisting of SEQ ID NO: 42 and SEQ ID NO: 79, or ananalog thereof having at least 90% sequence identity with the lightand/or heavy chain sequence.

According to some embodiments, the isolated monoclonal antibody orfragment thereof comprises a heavy chain variable region having thesequence set forth in SEQ ID NO: 34, and a light chain variable regionhaving the sequence set forth in SEQ ID NO: 42, SEQ ID NO: 49, SEQ IDNO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54;or an analog thereof having at least 90% sequence identity with thelight and/or heavy chain sequence. Each possibility represents aseparate embodiment of the invention

The invention also encompasses antibody or antibody fragment capable ofbinding with high affinity to an epitope within the human PVR protein towhich mAb 5B9 binds.

According to some embodiments, the isolated antibody or fragment thereofrecognizes human PVR with an affinity of at least 10⁻⁸M. According toother embodiments, an antibody or antibody fragment binds with anaffinity of 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M or evenhigher to human PVR. Each possibility represents a separate embodimentof the invention.

Analogs and derivatives of the isolated mAb antibodies, and the antibodyfragments described above, are also within the scope of the invention.In some embodiments, particular analogs or isolated mAbs or fragmentthereof comprising at least one variable region set forth in a sequenceselected from the group consisting of: SEQ ID NOs: 2, 10, 18, 26, 34 and42 are also within the scope of the present invention. Isolated mAbs orfragment thereof comprising at least one variable region set forth in asequence selected from the group consisting of: SEQ ID NOs: 69, 71, 73,75, 77 and 79 are also within the scope of the present invention.

According to some embodiments, the antibody or antibody fragment analoghave at least 90% sequence identity with the hypervariable region of thereference antibody sequence.

According to certain embodiments, the analog or derivative of theisolated antibody or fragment thereof has at least 91, 92, 93, 94, 95,96, 97, 98 or 99% sequence identity with a variable region of thereference antibody sequence. Each possibility represents a separateembodiment of the invention.

According to some embodiments, the antibody or antibody fragmentaccording to the invention comprises a heavy chain variable region setforth in SEQ ID NO: 2 or SEQ ID NO: 69, or an analog having at least 95%sequence similarity with said sequence. According to other embodiments,the antibody or antibody fragment according to the invention comprises aheavy chain variable region set forth in SEQ ID NO: 18 or SEQ ID NO: 73,or an analog having at least 95% sequence similarity with said sequence.According to other embodiments, the antibody or antibody fragmentaccording to the invention comprises a heavy chain variable region setforth in SEQ ID NO: 34 or SEQ ID NO: 77, or an analog having at least95% sequence similarity with said sequence.

According to some embodiments, the antibody or antibody fragmentcomprises a light chain variable region set forth in SEQ ID NO: 10 orSEQ ID NO: 71, or an analog having at least 95% sequence similarity withsaid sequence. According to other embodiments, the antibody or antibodyfragment comprises a light chain variable region set forth in SEQ ID NO:26 or SEQ ID NO: 75, or an analog having at least 95% sequencesimilarity with said sequence. According to other embodiments, theantibody or antibody fragment comprises a light chain variable regionset forth in SEQ ID NO: 42 or SEQ ID NO: 79, or an analog having atleast 95% sequence similarity with said sequence.

According to some embodiments, the antibody or antibody fragmentcomprises a heavy chain and a light chain, wherein: (i) the heavy chaincomprises SEQ ID NO: 2 and the light chain comprises SEQ ID NO: 10; (ii)the heavy chain comprises SEQ ID NO: 18 and the light chain comprisesSEQ ID NO: 26; or (iii) the heavy chain comprises SEQ ID NO: 34 and thelight chain comprises SEQ ID NO: 42. Analogs of the antibodies orfragments, having at least 95% sequence similarity with said heavy orlight chains are also included.

According to other embodiments, the antibody or antibody fragmentcomprises a heavy chain and a light chain, wherein: (i) the heavy chaincomprises SEQ ID NO: 69 and the light chain comprises SEQ ID NO: 71;(ii) the heavy chain comprises SEQ ID NO: 73 and the light chaincomprises SEQ ID NO: 75; or (iii) the heavy chain comprises SEQ ID NO:77 and the light chain comprises SEQ ID NO: 79. Analogs of theantibodies or fragments, having at least 95% sequence similarity withsaid heavy or light chains are also included.

According to some embodiments, the analog has at least 96, 97, 98 or 99%sequence identity with an antibody light or heavy chain variable regionsdescribed above. According to some embodiments, the analog comprises nomore than one amino acid substitution, deletion or addition to one ormore CDR sequences of the hypervariable region, namely, any one of theCDR sequences set forth in SEQ ID NOs: 4, 6, 8, 12, 14, 16, 20, 22, 24,28, 30, 32, 36, 38, 40, 44, 46, 48, 80, 81, 82, 83, 84, and 85. Eachpossibility represents a separate embodiment of the present invention.According to some embodiments, the amino acid substitution is aconservative substitution.

According to some embodiments, the antibody or antibody fragmentcomprises a hypervariable region (HVR) having light and heavy chainregions defined above, in which 1, 2, 3, 4, or 5 amino acids weresubstituted, deleted and/or added. Each possibility represents aseparate embodiment of the invention.

According to some embodiments, the antibody or antibody fragmentcomprises a HVR having light and heavy chain regions defined above, inwhich one amino acid was substituted. According to specific embodiments,the antibody or antibody fragment comprises a CDR as defined above, inwhich one amino acid was substituted. According to some specificembodiments, the antibody or antibody fragment comprises a light chainCDR2 as defined above, in which one amino acid was substituted.

According to some specific embodiments, the antibody or antibodyfragment comprises a light chain CDR2 having a sequence set forth in SEQID NO: 55 (WASSRHX), wherein X is selected from the group consisting ofA, R, D, E, P, and T. Each possibility represents a separate embodimentof the invention.

According to some embodiments, the antibody or antibody fragmentcomprises a light chain CDR2 having a sequence selected from the groupconsisting of SEQ ID NO: 56-61.

According to some embodiments, the isolated monoclonal antibody or theantibody fragment comprises a CDR set selected from the group consistingof:

-   -   i. a CDR set of six CDRs wherein: HC CDR1 is selected from        GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); HC CDR2        is EIFPGSGRINFNEKFKG (SEQ ID NO: 38); HC CDR3 is TKIYGNSFDY (SEQ        ID NO: 40); LC CDR1 is selected from KASQDVGTAVV (SEQ ID NO: 44)        and KASQDVGTAV (SEQ ID NO: 85); LC CDR2 is selected from the        group consisting of: WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID        NO: 56), WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58),        WASSRHE (SEQ ID NO: 59), WASSRHP (SEQ ID NO: 60), and WASSRHT        (SEQ ID NO: 61); and LC CDR3 is QQYSRYPLT (SEQ ID NO: 48).    -   ii. a CDR set of six CDRs wherein: HC CDR1 sequence is selected        from GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); HC CDR2        is selected from EIHPDSSKINYTPSQ (SEQ ID NO: 6) and        EIHPDSSKINYTPSQKD (SEQ ID NO: 81); HC CDR3 is selected from        PDGNYNALDYW (SEQ ID NO: 8) and PDGNYNALDY (SEQ ID NO: 82); LC        CDR1 is KASQDVGTAVT (SEQ ID NO: 12); LC CDR2 is WASTRHT (SEQ ID        NO: 14); and LC CDR3 is QQYSRYPYT (SEQ ID NO: 16).    -   iii. a CDR set of six CDRs wherein: HC CDR1 sequence is selected        from the GYTFTEYTMH (SEQ ID NO: 20) and EYTMH (SEQ ID NO: 83);        HC CDR2 is GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); HC CDR3 is VIPLEY        (SEQ ID NO: 24); LC CDR1 is KASQNVYTNVA (SEQ ID NO: 28); LC CDR2        is SASYRYR (SEQ ID NO: 30); and LC CDR3 is QQYNSYPLA (SEQ ID NO:        32).

The present invention thus provides a monoclonal antibody thatspecifically binds the human protein PVR, or a binding fragment thereof,wherein said monoclonal antibody or fragment comprises a set of six CDRsequences wherein the set is selected from the group consisting of:

-   -   i. SEQ ID NOs. 4, 6, 8, 12, 14 and 16;    -   ii. SEQ ID NOs. 20, 22, 24, 28, 30 and 32;    -   iii. SEQ ID NOs. 36, 38, 40, 44, 46 and 48;    -   iv. SEQ ID NOs. 36, 38, 40, 44, 55 and 48;    -   v. SEQ ID NOs. 80, 81, 82, 12, 14 and 16;    -   vi. SEQ ID NOs. 83, 22, 24, 28, 30 and 32;    -   vii. SEQ ID NOs. 84, 38, 40, 85, 46 and 48; and    -   viii. SEQ ID NOs. 84, 38, 40, 85, 55 and 48.

The present invention also provides monoclonal antibodies and bindingfragments thereof, comprising a heavy chain and a light chain, whereinsaid chains comprises a set of heavy chain variable region sequence andlight chain variable region sequence, said set is selected from thegroup consisting of:

-   -   i. SEQ ID NOs: 2 and 10;    -   ii. SEQ ID NOs: 69 and 71;    -   iii. SEQ ID NOs: 18 and 26;    -   iv. SEQ ID NOs: 73 and 75;    -   v. SEQ ID NOs: 34 and 42; and    -   vi. SEQ ID NOs: 77 and 79.

According to some embodiments, the antibody or antibody fragment iscapable of inhibiting human PVR binding to TIGIT expressed on T cells orNK cells.

According to a specific embodiment, the mAb is selected from the groupconsisting of: chimeric antibody, and an antibody fragment comprising atleast the antigen-binding portion of an antibody. According to specificembodiments, the antibody is a chimeric antibody. According to yet otherembodiments, the chimeric antibody comprised human constant region.According to yet other embodiments, the chimeric monoclonal antibodycomprises human IgG1 constant region. According to a specificembodiment, the antibody fragment is selected from the group consistingof: Fab, Fab′, F(ab′)₂, Fd, Fd′, Fv, dAb, isolated CDR region, singlechain antibody (scab), “diabodies”, and “linear antibodies”. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, the antibody or antibody fragmentcomprises a framework sequence selected from the group consisting of:mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, humanIgG3, and human IgG4. Each possibility represents a separate embodimentof the present invention.

According to some embodiments, a conjugate comprising the antibody orfragment thereof as described above is provided.

According to some embodiments, the conjugate comprises a carrierprotein.

Polynucleotide sequences encoding monoclonal antibodies, having highaffinity and specificity for PVR, as well as vectors and host cellscarrying these polynucleotide sequences, are provided according toanother aspect of the present invention.

According to some embodiments, polynucleotide sequences encoding theamino acid sequences of HC variable region and light LC variable regiondescribed above are provided.

According to some embodiments, the polynucleotide sequence encodes anantibody or antibody fragment or chain capable of binding to an epitopewithin the human PVR protein to which binds: (i) a monoclonal antibody(herein identified as 4E5) having a heavy chain variable region of SEQID NO: 2 and a light chain variable region of SEQ ID NO: 10; (ii) amonoclonal antibody (herein identified as 7D4) having a heavy chainvariable region of SEQ ID NO:18 and a light chain variable region of SEQID NO: 26; or (iii) a monoclonal antibody (herein identified as 5B9)having a heavy chain variable region of SEQ ID NO: 34 and a light chainvariable region of SEQ ID NO: 42.

According to some embodiments, the polynucleotide sequence encodes anantibody or antibody fragment or chain comprising the sequence set forthin SEQ ID NO: 2 or SEQ ID NO: 69. According to some embodiments, thepolynucleotide sequence encodes an antibody or antibody fragment orchain comprising the sequence set forth in SEQ ID NO: 10 or SEQ ID NO:71.

According to other embodiments, the polynucleotide sequence encodes anantibody or antibody fragment or chain comprising the sequence set forthin SEQ ID NO: 18 or SEQ ID NO: 73. According to additional embodiments,the polynucleotide sequence encodes an antibody or antibody fragment orchain comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:75.

According to other embodiments, the polynucleotide sequence encodes anantibody or antibody fragment or chain comprising the sequence set forthin SEQ ID NO: 34 or SEQ ID NO: 77. According to additional embodiments,the polynucleotide sequence encodes an antibody or antibody fragment orchain comprising the sequence set forth in SEQ ID NO: 42 or SEQ ID NO:79.

According to yet some embodiments, the polynucleotide sequence accordingto the invention encodes an antibody or antibody fragment or chaincomprising the six CDR sequences: (i) heavy chain CDR1 having thesequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), heavy chainCDR2 having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chainCDR3 having the sequence: PDGNYNALDY (SEQ ID NO: 82), light chain CDR1having the sequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2having the sequence: WASTRHT (SEQ ID NO: 14), and light chain CDR3having the sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1having the sequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 having thesequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 having thesequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 having the sequence:KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 having the sequence:SASYRYR (SEQ ID NO: 30), and light chain CDR3 having the sequence:QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 having the sequenceSNYWIE (SEQ ID NO: 84), heavy chain CDR2 having the sequence:EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 having the sequence:TKIYGNSFDY (SEQ ID NO: 40), light chain CDR1 having the sequence:KASQDVGTAV (SEQ ID NO: 85), light chain CDR2 having the sequence:WASSRHN (SEQ ID NO: 46), and light chain CDR3 having the sequence:QQYSRYPLT (SEQ ID NO: 48).

According to some embodiments, the polynucleotide sequences definedabove encodes a molecule selected from the group consisting of: anantibody, an antibody fragment comprising at least an antigen-bindingportion, and an antibody conjugate comprising said antibody or antibodyfragment. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, the polynucleotide sequence encoding amonoclonal antibody heavy chain variable region, comprises a sequenceset forth in SEQ ID NO: 1 or SEQ ID NO: 68, or a variant thereof havingat least 90% sequence identity.

According to some embodiments, the polynucleotide sequence encoding amonoclonal antibody heavy chain variable region, comprises a sequenceset forth in SEQ ID NO: 17 or SEQ ID NO: 72, or a variant thereof havingat least 90% sequence identity.

According to some embodiments, the polynucleotide sequence encoding amonoclonal antibody heavy chain variable region, comprises a sequenceset forth in SEQ ID NO: 33 or SEQ ID NO: 76, or a variant thereof havingat least 90% sequence identity.

According to some embodiments, the polynucleotide sequence encoding amonoclonal antibody light chain variable region, comprises a sequenceset forth in SEQ ID NO: 9 or SEQ ID NO: 70, or a variant thereof havingat least 90% sequence identity.

According to some embodiments, the polynucleotide sequence encoding amonoclonal antibody light chain variable region, comprises a sequenceset forth in SEQ ID NO: 25 or SEQ ID NO: 74, or a variant thereof havingat least 90% sequence identity.

According to some embodiments, the polynucleotide sequence encoding amonoclonal antibody light chain variable region, comprises a sequenceset forth in SEQ ID NO: 41 or SEQ ID NO: 78, or a variant thereof havingat least 90% sequence identity.

The present invention provides, according to some embodiments, apolypeptide comprising at least one sequence encoded by at least onepolynucleotide sequence disclosed above.

In a further aspect, the present invention provides a nucleic acidconstruct comprising a nucleic acid molecule encoding at least oneantibody chain or fragment thereof according to the present invention.According to some embodiments the nucleic acid construct is a plasmid.

According to some embodiments the plasmid comprises a polynucleotidesequence set forth in SEQ ID NO: 1, SEQ ID NO: 17, or SEQ ID NO: 33.

According to some embodiments the plasmid comprises a polynucleotidesequence set forth in SEQ ID NO: 68, SEQ ID NO: 72, or SEQ ID NO: 76.

According to other embodiments the plasmid comprises a polynucleotidesequence set forth in SEQ ID NO: 9, SEQ ID NO: 25, or SEQ ID NO: 41.

According to other embodiments the plasmid comprises a polynucleotidesequence set forth in SEQ ID NO: 70, SEQ ID NO: 74, or SEQ ID NO: 78.

In still another aspect the present invention provides a hybridoma cellcapable of producing an antibody or an antibody fragment comprising thespecific CDR sequences and/or specific heavy and light chain variableregions defined above.

According to some embodiments, a hybridoma cell is provided comprisingat least one polynucleotide sequence disclosed above.

According to some embodiments, the hybridoma is a cable of producing amonoclonal antibody comprising the six complementarity determiningregions (CDRs) sequences: (i) heavy chain CDR1 having the sequence:GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), heavy chain CDR2having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3having the sequence: PDGNYNALDY (SEQ ID NO: 82), light chain CDR1 havingthe sequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 having thesequence: WASTRHT (SEQ ID NO: 14), and light chain CDR3 having thesequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 having thesequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 having the sequence:GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 having the sequence:VIPLEY (SEQ ID NO: 24), light chain CDR1 having the sequence:KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 having the sequence:SASYRYR (SEQ ID NO: 30), and light chain CDR3 having the sequence:QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 having the sequenceSNYWIE (SEQ ID NO: 84), heavy chain CDR2 having the sequence:EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 having the sequence:TKIYGNSFDY (SEQ ID NO: 40), light chain CDR1 having the sequence:KASQDVGTAV (SEQ ID NO: 85), light chain CDR2 having the sequence:WASSRHN (SEQ ID NO: 46), and light chain CDR3 having the sequence:QQYSRYPLT (SEQ ID NO: 48).

Antibodies or fragments thereof according to the present invention maybe attached to a cytotoxic moiety, a radioactive moiety, or anidentifiable moiety.

The present invention provides, according to another aspect, apharmaceutical composition comprising as an active ingredient, at leastone antibody, antibody fragment or conjugates thereof, that recognizesPVR with high affinity and specificity, and optionally at least onepharmaceutical acceptable excipient, diluent, salt or carrier.

According to some embodiments, the pharmaceutical composition comprisesa monoclonal antibody or a fragment thereof which is capable of bindingto an epitope within the human PVR protein to which binds a monoclonalantibody selected from the group consisting of: (i) an antibody hereinidentified as 4E5 (also denoted PVR.07), having a heavy chain variableregion of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO:10; (ii) an antibody herein identified as 7D4 (also denoted PVR.01)having a heavy chain variable region of SEQ ID NO: 18 and a light chainvariable region of SEQ ID NO: 26; and (ii) an antibody herein identifiedas 5B9 (also denoted PVR.09) having a heavy chain variable region of SEQID NO: 34 and a light chain variable region of SEQ ID NO: 42.

According to some embodiments, the pharmaceutical composition comprisesa monoclonal antibody or antibody fragment thereof comprising the sixCDRs: (i) heavy chain CDR1 having the sequence: GFDFSRYW (SEQ ID NO: 4)or RYWMT (SEQ ID NO: 80, heavy chain CDR2 having the sequence:EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3 having the sequence:PDGNYNALDY (SEQ ID NO: 82), light chain CDR1 having the sequence:KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 having the sequence:WASTRHT (SEQ ID NO: 14), and light chain CDR3 having the sequence:QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 having the sequenceEYTMH (SEQ ID NO: 83), heavy chain CDR2 having the sequence:GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 having the sequence:VIPLEY (SEQ ID NO: 24), light chain CDR1 having the sequence:KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 having the sequence:SASYRYR (SEQ ID NO: 30), and light chain CDR3 having the sequence:QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 having the sequenceSNYWIE (SEQ ID NO: 84), heavy chain CDR2 having the sequence:EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 having the sequence:TKIYGNSFDY (SEQ ID NO: 40), light chain CDR1 having the sequence:KASQDVGTAV (SEQ ID NO: 85), light chain CDR2 having the sequence:WASSRHN (SEQ ID NO: 46), and light chain CDR3 having the sequence:QQYSRYPLT (SEQ ID NO: 48).

According to some embodiments, the pharmaceutical composition comprisesa monoclonal antibody or fragment thereof comprising a heavy chainvariable region having a sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 69, SEQ ID NO: 18, SEQ ID NO: 73, SEQ ID NO:34, and SEQ ID NO: 77. Each possibility represent a separate embodimentof the invention

According to some embodiments, the pharmaceutical composition comprisesa monoclonal antibody or fragment thereof comprising a light chainvariable region having a sequence selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 71, SEQ ID NO: 26, SEQ ID NO: 75, SEQ ID NO:42, and SEQ ID NO: 79. Each possibility represents a separate embodimentof the invention.

According to a specific embodiment, the pharmaceutical compositioncomprises a monoclonal antibody or fragment thereof comprising a heavychain variable region having the sequence set forth in SEQ ID NO: 2 orSEQ ID NO: 69 and a light chain variable region having the sequence setforth in SEQ ID NO: 10 or SEQ ID NO: 71.

According to a specific embodiment, the pharmaceutical compositioncomprises a monoclonal antibody or fragment thereof comprising a heavychain variable region having the sequence set forth in SEQ ID NO: 18 orSEQ ID NO: 73 and a light chain variable region having the sequence setforth in SEQ ID NO: 26 or DES ID NO: 75.

According to a specific embodiment, the pharmaceutical compositioncomprises a monoclonal antibody or fragment thereof comprising a heavychain variable region having the sequence set forth in SEQ ID NO: 34 orSEQ ID NO: 77 and a light chain variable region having the sequence setforth in SEQ ID NO: 42 or SEQ ID NO: 79.

According to some embodiments, the pharmaceutical composition comprisesa combination of at least two antibodies, or antibody fragments, whichrecognizes human PVR.

According to yet other embodiments, the pharmaceutical compositioncomprises one mAb or fragment that specifically binds PVR according tothe invention, and one mAb or fragment that specifically binds adifferent antigen, such as, cell-receptor, tumor antigen orimmunomodulatory protein.

Also provided are pharmaceutical compositions, comprising at least oneantibody, antibody fragment or antibody conjugate according to theinvention, for use in restoring NK cytotoxicity by inhibiting binding ofPVR to TIGIT expressed on NK cells.

According to some embodiments, the antibody, antibody fragment orantibody conjugate is capable of inhibiting human PVR binding to TIGITexpressed on T-cells.

According to some embodiments, the pharmaceutical composition accordingto the present invention is for use in cancer immunotherapy or inenhancing immune response.

The cancer can be any cancer that express PVR. According to someembodiments, the cancer overexpresses PVR.

According to some embodiments of the invention, the cancer is ametastatic cancer. According to some embodiments, the pharmaceuticalcomposition according to the present invention is for use in inhibitingformation or distribution of metastases or reducing the total number ofmetastases in a subject.

According to some embodiments of the invention, the cancer is selectedfrom the group consisting of a melanoma, a breast cancer, an ovariancancer, a pancreatic cancer, a colorectal cancer, a colon cancer, acervical cancer, a kidney cancer, a lung cancer, a thyroid cancer, aprostate cancer, a brain cancer, a renal cancer, a throat cancer, alaryngeal carcinoma, a bladder cancer, a hepatic cancer, a fibrosarcoma,an endometrial cells cancer, a glioblastoma, sarcoma, a myeloid, aleukemia and a lymphoma. Each possibility represents a separateembodiment of the invention.

According to some embodiments, the cancer is a solid cancer. Accordingto some specific embodiments, the solid cancer is selected from thegroup consisting of melanoma (skin), lung, colon, breast, uterine, andrenal cancer. According to specific embodiments, the cancer is selectedfrom the group consisting of breast cancer, lung cancer, andliposarcoma.

According to other embodiments, the cancer is hematologic cancer.According to some embodiments, the hematologic cancer is myeloidleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,myeloproliferative diseases, multiple myeloma, or myelodysplasticsyndrome. Each possibility represents a separate embodiment of theinvention. According to certain embodiments, the cancer is leukemia.According to specific embodiments, the cancer is acute myeloid leukemia(AML).

According to some embodiments, the pharmaceutical composition accordingto the present invention is for use in treating a viral infection.

According to some embodiments, the viral infection is caused by a viruswhich binds a target cell via a PVR on a surface of the infected cell.

According to some embodiments, the pharmaceutical composition accordingto the present invention is for use in treating an angiogenesis-relateddisease or disorder. According to certain embodiments, theangiogenesis-related disease or disorder is selected from the groupconsisting of: cancer, cell proliferative diseases of the eye (oculardiseases), retinal disorders, and inflammatory disease. Each possibilityrepresents a separate embodiment of the invention.

According to yet another aspect, the present invention provides a methodof inhibiting binding of human PVR to at least one ligand by using amonoclonal antibody or antibody fragment defined above.

According to an additional aspect, the present invention provides amethod for enhancing immune response in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of a monoclonal antibody, antibody fragment or antibody conjugatedefined above.

According to yet another aspect, the present invention provides a methodof treating cancer comprising administering to a subject in needthereof, a therapeutically effective amount of a pharmaceuticalcomposition comprising at least one antibody, antibody fragment orconjugate thereof, that recognizes human PVR with high affinity andspecificity and capable of inhibiting its binding to its ligand.

According to some embodiments, the antibody in the pharmaceuticalcomposition administered is selected from the group consisting of: (i) amonoclonal antibody comprising the CDR sequences contained in heavychain variable region set forth in SEQ ID NO: 2 and the CDR sequencescontained in light chain variable region set forth in SEQ ID NO: 10;(ii) a monoclonal antibody comprising the CDR sequences contained inheavy chain variable region set forth in SEQ ID NO: 18 and the CDRsequences contained in light chain variable region set forth in SEQ IDNO: 26; or (iii) a monoclonal antibody comprising the CDR sequencescontained in heavy chain variable region set forth in SEQ ID NO: 34 andthe CDR sequences contained in light chain variable region set forth inSEQ ID NO: 42.

According to some specific embodiments, the monoclonal antibody in thepharmaceutical composition administered comprises: heavy chain CDR1comprises the sequence selected from the group consisting of GFDFSRYW(SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); heavy chain CDR2 comprises thesequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); heavy chain CDR3 comprises thesequence: PDGNYNALDY (SEQ ID NO: 82); light chain CDR1 comprises thesequence: KASQDVGTAVT (SEQ ID NO: 12); light chain CDR2 comprises thesequence: WASTRHT (SEQ ID NO: 14); and light chain CDR3 comprises thesequence: QQYSRYPYT (SEQ ID NO: 16). Each possibility represents aseparate embodiment of the invention.

According to other specific embodiments, the monoclonal antibody in thepharmaceutical composition administered comprises: heavy chain CDR1comprises the sequence EYTMH (SEQ ID NO: 83); heavy chain CDR2 comprisesthe sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); heavy chain CDR3comprises the sequence: VIPLEY (SEQ ID NO: 24); light chain CDR1comprises the sequence: KASQNVYTNVA (SEQ ID NO: 28); light chain CDR2comprises the sequence: SASYRYR (SEQ ID NO: 30); and light chain CDR3comprises the sequence: QQYNSYPLA (SEQ ID NO: 32).

According to other specific embodiments, the monoclonal antibody in thepharmaceutical composition administered comprises: heavy chain CDR1comprises the sequence SNYWIE (SEQ ID NO: 84); heavy chain CDR2comprises the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38); heavy chainCDR3 comprises the sequence: TKIYGNSFDY (SEQ ID NO: 40); light chainCDR1 comprises the sequence: KASQDVGTAV (SEQ ID NO: 85); light chainCDR2 comprises the sequence: WASSRHN (SEQ ID NO: 46); and light chainCDR3 comprises the sequence: QQYSRYPLT (SEQ ID NO: 48).

According to some embodiments of the invention, the therapeuticallyeffective amount results in a decrease in tumor size or in the number ofmetastases in the subject.

According to some embodiments, the method of treating cancer comprisesadministering or performing at least one additional anti-cancer therapy.According to certain embodiments, the additional anticancer therapy issurgery, chemotherapy, radiotherapy, or immunotherapy.

According to some embodiments, the method of treating cancer comprisesadministration of a monoclonal antibody that recognizes human PVR withhigh affinity and specificity and an additional anti-cancer agent.According to some embodiments, the additional anti-cancer agent isselected from the group consisting of: immune-modulator, activatedlymphocyte cell, kinase inhibitor and chemotherapeutic agent.

According to other embodiments, the additional immune-modulator is anantibody, antibody fragment or antibody conjugate that binds to anantigen other than human PVR.

According to some embodiments, the additional immune-modulator is anantibody against an immune checkpoint molecule. According to someembodiments, the additional immune modulator is an antibody against animmune checkpoint molecule selected from the group consisting of humanprogrammed cell death protein 1 (PD-1), PD-L1 and PD-L2,carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1),lymphocyte activation gene 3 (LAG3), CD137, OX40 (also referred to asCD134), killer cell immunoglobulin-like receptors (KIR), TIGIT and anycombination thereof. Each possibility represents a separate embodimentof the invention.

According to some embodiments, the anti-cancer agent is selected fromthe group consisting of: Erbitux, cytarabine, fludarabine, fluorouracil,mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine,vinblastine, vinorelbine, carmustine, lomustine, chlorambucil,cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine,melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin,doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide,teniposide and any combination thereof. Each possibility represents aseparate embodiment of the invention.

According to some embodiments, the anti-cancer agent is epidermal growthfactor receptor (EGFR) inhibitor. According to some embodiments, theEGFR inhibitor is selected from the group consisting of: Cetuximab(Erbitux®), Panitumumab (Vectibix®), and necitumumab (Portrazza®).According to some embodiments, the EGFR inhibitor is Cetuximab(Erbitux®).

According to some embodiments of the invention, the subject is a humansubject.

According to some embodiments of the invention, the use furthercomprises the use of an agent that downregulates the activity orexpression of an immune co-inhibitory receptor.

According to some embodiments of the invention, the immune cell is a Tcell.

According to some embodiments of the invention, the immune co-inhibitoryreceptor is selected from the group consisting of PD-1, TIGIT, DNAM-1,CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1, SIGLEC10, and2B4. Each possibility represents a separate embodiment of the invention.

According to an aspect, the present invention provides a method formodulating immune system function and/or activity comprising modulatingthe binding of PVR to TIGIT using an antibody according to theinvention.

According to an aspect, the present invention provides a method ofpreventing or treating a viral infection of a virus that utilizes CD155as an entry receptor, in a subject in need thereof, the method comprisesadministering to the subject a therapeutically effective amount of ananti PVR monoclonal antibody described herein. According to someembodiments the virus is selected from the group consisting of: poliovirus, coxsackie virus, adeno virus and human deficiency virus (HIV).

According to yet another aspect, the present invention provides a methodfor treating an angiogenesis-related disease or disorder. According tocertain embodiments, the angiogenesis-related disease or disorder isselected from the group consisting of: cancer, cell proliferativediseases of the eye (ocular diseases), retinal disorders, andinflammatory disease. Each possibility represents a separate embodimentof the invention.

According to some embodiments, the method of treating cancer involvespreventing or reducing formation, growth or spread of metastases in asubject by inhibiting angiogenesis.

According to an aspect, the present invention provides a method ofdiagnosing or prognosing cancer or infectious disease in a subject, themethod comprises determining the expression level of PVR in a biologicalsample of said subject using at least one antibody as described herein.

The present invention further comprises, according to another aspect, amethod of determining or quantifying the expression of PVR, the methodcomprising contacting a biological sample with an antibody or antibodyfragment, and measuring the level of complex formation, wherein theantibody or antibody fragment comprises the complementarity determiningregions (CDRs) selected from the group consisting of: (i) heavy chainCDR1 having the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO:80), heavy chain CDR2 having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO:6), heavy chain CDR3 having the sequence: PDGNYNALDY (SEQ ID NO: 82),light chain CDR1 having the sequence: KASQDVGTAVT (SEQ ID NO: 12), lightchain CDR2 having the sequence: WASTRHT (SEQ ID NO: 14), and light chainCDR3 having the sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chainCDR1 having the sequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 havingthe sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 havingthe sequence: VIPLEY (SEQ ID NO: 24), light chain CDR1 having thesequence: KASQNVYTNVA (SEQ ID NO: 28), light chain CDR2 having thesequence: SASYRYR (SEQ ID NO: 30), and light chain CDR3 having thesequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) heavy chain CDR1 havingthe sequence SNYWIE (SEQ ID NO: 84), heavy chain CDR2 having thesequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), heavy chain CDR3 having thesequence: TKIYGNSFDY (SEQ ID NO: 40), light chain CDR1 having thesequence: KASQDVGTAV (SEQ ID NO: 85), light chain CDR2 having thesequence: WASSRHN (SEQ ID NO: 46), and light chain CDR3 having thesequence: QQYSRYPLT (SEQ ID NO: 48).

Determining and quantifying methods may be performed in-vitro or ex-vivoaccording to some embodiments or may be used in diagnosing conditionsassociated with expression of PVR. The antibodies according to thepresent invention may be also used to configure screening methods. Forexample, an enzyme-linked immunosorbent assay (ELISA), or aradioimmunoassay (RIA) can be constructed for measuring levels ofsecreted or cell-associated polypeptide using the antibodies and methodsknown in the art.

According to some embodiments, the method for detecting or quantifyingthe presence of PVR comprises the steps of:

-   -   i. incubating a sample with an antibody specific to PVR or an        antibody fragment thereof comprising at least an antigen-binding        portion;    -   ii. detecting the bound PVR using a detectable probe.

According to some embodiments, the method further comprises the stepsof:

-   -   iii. comparing the amount of (ii) to a standard curve obtained        from a reference sample containing a known amount of PVR; and    -   iv. calculating the amount of the PVR in the sample from the        standard curve.

According to some particular embodiments the sample is a body fluid.

According to some embodiments, the method is performed in-vitro orex-vivo.

A kit for measuring the expression of PVR in biological sample is alsoprovided comprising at least one antibody or antibody fragmentcomprising the complementarity determining regions (CDRs) selected fromthe group consisting of: (i) heavy chain CDR1 having the sequence:GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), heavy chain CDR2having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), heavy chain CDR3having the sequence: PDGNYNALDY (SEQ ID NO: 82), light chain CDR1 havingthe sequence: KASQDVGTAVT (SEQ ID NO: 12), light chain CDR2 having thesequence: WASTRHT (SEQ ID NO: 14), and light chain CDR3 having thesequence: QQYSRYPYT (SEQ ID NO: 16); (ii) heavy chain CDR1 having thesequence EYTMH (SEQ ID NO: 83), heavy chain CDR2 having the sequence:GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), heavy chain CDR3 having the sequence:VIPLEY (SEQ ID NO:24), light chain CDR1 having the sequence: KASQNVYTNVA(SEQ ID NO: 28), light chain CDR2 having the sequence: SASYRYR (SEQ IDNO: 30), and light chain CDR3 having the sequence: QQYNSYPLA (SEQ ID NO:32); or (iii) heavy chain CDR1 having the sequence SNYWIE (SEQ ID NO:84), heavy chain CDR2 having the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO:38), heavy chain CDR3 having the sequence: TKIYGNSFDY (SEQ ID NO: 40),light chain CDR1 having the sequence: KASQDVGTAV (SEQ ID NO: 85), lightchain CDR2 having the sequence: WASSRHN (SEQ ID NO: 46), and light chainCDR3 having the sequence: QQYSRYPLT (SEQ ID NO: 48).

According to an aspect, the present invention provides a kit fordetecting cancer, the diagnostic kit comprises an antibody of fragmentthereof as disclosed herein.

According to some embodiments, the invention provides a method ofdiagnosing, assessing the severity or staging an immune-related diseaseor a proliferative disease comprising determining the expression oractivity of PVR in a sample from a subject using an antibody accordingto the present invention or a fragment or conjugate thereof, andcomparing the expression or activity of PVR to a reference amount of PVRexpression or activity. Said reference amount may be obtained from asample taken from a normal subject, from the same subject while being ina different stage of the disease or is determined from clinical data ofa large population of subjects.

Further embodiments and the full scope of applicability of the presentinvention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-IC are graphs depicting the correlation of PVR mRNA expressionlevels (high or low as indicated) with survival probability. Thecorrelation was measured for lung cancer (FIG. 1A), breast cancer (FIG.1B) and liposarcoma (FIG. 1C). Data sets of mRNA expression wereobtained from the GEO site and analyzed using bioprofiling.de site, asfollows: for lung cancer GEO dataset ID: GSE31210, for breast cancer GEOdataset ID: GSE25055 and for liposarcoma GEO dataset ID: GSE30929.

FIG. 2 is a schematic illustration of receptor expression on immune andtumor cells. TIGIT relates to a co-inhibitory receptor on many immunecells (e.g. T cells); DNAM-1 (also termed CD226) relates to anactivating receptor on many immune cells (e.g. T cells); Fc Receptorrelates to the strong activating receptor expressed mainly on NK cellsbut also on myeloid cells including neutrophils and macrophages; PVRrelates to an inhibitory ligand for TIGIT (weaker binding to DNAM-1),expressed by many tumor cells; Nectin-2 relates to an activating ligandfor DNAM-1 (marginal recognition by TIGIT), expressed by many tumorcells. Binding of anti-PVR according to the present invention shows adual effect: 1) enhancing the killing of tumor cells via Fc receptors;and 2) increasing activation of immune cells by blocking the interactionwith TIGIT.

FIGS. 3A-3D. are graphs depicting FACS analysis of the four anti-PVRantibodies generated. Shown is the efficacy of the antibodies inblocking the direct binding of TIGIT-Fc to a tumor cell line. FIG. 3Aillustrates a non-blocking anti-PVR antibody (anti-PVR mAb antibody 2G3,also termed hPVR.17). FIGS. 3B-3D show that three of the antibodiesgenerated, namely 5B9 (also termed hPVR.09), 7D4 (also termed hPVR.01)and 4E5 (also termed hPVR.07), respectively, are anti-PVR blocking mAbsas shown by blockage of TIGIT-Ig binding. Hybridomas soups (5 μl of/well) were added to HepG2 cells. TIGIT-Fc was used at 2 μg/well tofinal concentration of 20 μg/ml and levels of cell bound TIGIT weremeasured by FACS after adding fluorescently labeled anti-Fc Ab. Filledhistograms depict background staining by the anti-Fc reagent.

FIG. 4 is a graph that shows how blocking of PVR-TIGIT interactions withthe anti-PVR mAb antibody 7D4 (also termed hPVR.01) enhances NK cellkilling of the human cell line MDA-MB-231 (Breast Adenocarcinoma).Specific killing is calculated based on the secretion of[35S]—Methionine from the target cells. Control (Ctrl) is non-relatedmouse IgG. P Value=0.0056.

FIG. 5 is a graph depicting that blocking of PVR-TIGIT interactionsusing the anti-PVR mAb 4E5 (also termed hPVR.07) enhances NK cellkilling of human cancer cell line HepG2 (hepatocellular carcinoma).No—killing of HepG2 without mAb; anti-PVR 4E5—killing of HepG2 withmouse anti-PVR 4E5mIgG1 (no activation of human Fc receptor); anti-PVR4e5hIgG1-killing of HepG2 with anti-PVR 4E5-hIgG1 (activation of humanFc receptor), Erbitux (P.C)—a positive control mAb Erbitux (anti-EGFR).All mAbs were used at 10 μg/ml. P values: anti-PVR 4E5—0.04, anti-PVR4e5hIgG1—0.000746 and Erbitux (positive control)—0.003219.

FIGS. 6A-60 are graphs depicting that human tumor cell lines express PVRand Nectin-2. Melanoma cells (FIGS. 6A-E), breast cancer cells (FIGS.6F-H), colorectal cells (FIG. 6I), kidney cells (FIG. 6J), lung cancercells (FIG. 6K), prostate cancer cells (FIG. 6L), brain tumor cells(FIG. 6M), and hepatocellular carcinoma cells (FIGS. 6N-O) all expressPVR and Nectin-2. mAbs were used at 0.2 μg/well: a commercialanti-Nectin-2 mAb and the anti-PVR mAb 4E5 (also termed hPVR.07).

FIGS. 7A-7D are graphs of FACS analysis depicting that PVR is the mainTIGIT ligand. FIG. 7A illustrates that HepG2 cells (human hepatocellularcarcinoma cells) express PVR and Nectin-2. FIG. 7B illustrates thatpurified anti-PVR mAb 4E5 (also termed hPVR.07) (0.15 μg) almostcompletely (above 97%) blocks TIGIT-Ig (2 μg/ml) binding, despite thefact that these cells also express Nectin-2. FIG. 7C illustrates thatCHO cells express high levels of hNectin-2. FIG. 7D shows lack ofstaining of the same CHO cells as in FIG. 7C by all PVR mAbs, meaningthat there is no direct recognition of Nectin-2 by anti-PVR mAbs, andthus the blocking of TIGIT binding seen in FIG. 7B cannot be explainedby blocking of Nectin-2 but rather it is the result of a direct PVRblocking.

FIGS. 8A-8C depict a similar binding efficacy of all of the anti-PVRmAbs to human PVR. All three blocking clones generate similar (less than10% difference) binding to both endogenous PVR HepG2 cells (FIG. 8A) andoverexpressed hPVR B16-hPVR cells (FIG. 8B). Binding was also examinedusing Vero cell line from African green monkey (FIG. 8C) which express aPVR protein with 93% similarity to human PVR. In this case the differentAbs showed differential staining intensities. 0.2 μg of each mAb used inall cases.

FIG. 9 is a graph depicting that the anti-PVR antibodies of theinvention do not recognize canine PVR. Human TIGIT-Fc (10 μg/ml) isstrongly cross-reactive and binds to the canine MDCK cell line. None ofthe PVR mAbs were able to bind to these cells suggesting that do notrecognize the canine PVR.

FIGS. 10A-10D depict that Nectin-2 is preferentially bound by DNAM-1 andnot by TIGIT. Cells over expressing either PVR or Nectin2 were stainedusing the indicated antibody concentrations. FIG. 10A illustratesbinding of TIGIT-Fc to Nectin2 expressing cells; FIG. 10B illustratesbinding of DNAM-FC to Nectin-2 expressing cells. FIG. 10C illustratesbinding of TIGIT to PVR expressing cells. FIG. 10D illustrates bindingof DNAM-1 to PVR expressing cells.

FIG. 11 shows the effect of anti-PVR antibodies on T cell proliferation.Human PBMCs were CFSE labeled and incubated with target cells in thepresence of the indicated antibodies. Results are presented as foldincreased proliferation relative to the control. The results are ofpooled 7 experiments total of 10 healthy donors. P values: for mAb4E5—0.000241, for mAb 5B9—1.96E⁻⁰⁵, for anti PD-1-0.016303, for antiCTLA4—0.000171, and for 4E5hIgG1—0.008176.

FIG. 12 shows the combined effect of anti-PVR antibodies and otherantibodies on T cell proliferation. Human PBMCs were CFSE labeled andincubated with target cells in the presence of the indicated combinationof antibodies. Results are presented as fold increased proliferationrelative to the control. The results are 7 independent experiments using12 healthy donors. P. Values: anti PD1+anti CTLA—47.54E⁻⁰³, antiPD-1+4E5—7.02E⁻⁰², anti PD-1+5B9—1.11E⁻⁰⁴, anti CTLA4+4E5—1.37E⁻⁰³, antiCTLA4+5B9—5.47E⁻⁰⁶.

FIG. 13 depicts the specific effect of anti-PVR antibodies on CD8 T cellproliferation. Human PBMCs were CFSE labeled and incubated with targetcells (A549) in the presence of the indicated antibodies. CD8 positivecells were count by FACS after 9-12 days in culture. Results arepresented as fold increased proliferation relative to the control. Theresults are of 2 independent experiments using healthy PBMC donors.

FIG. 14 depicts the ratio of CD8 cells to CD4 cells following theinduction with different antibodies. Human PBMCs were CFSE labeled andincubated with target cells (A549) in the presence of the indicatedantibodies. Cells were counted after 9-12 days in culture. The resultsare of pooled 2 experiments total of 2 healthy donors.

FIG. 15 shows the effect of anti-PVR antibodies on NK degranulation.Human NK cells were incubated with MDA-MB-231 cells (triple negativebreast cancer cell line) in the presence of the indicated antibodies.The results are representative of 7 independent experiments done with 5different healthy NK cell donors. P values: PVR4E5—0.005063,PVR5B9—0.00374, PVR7D4—0.019448, PVR4E5-hIgG1—2.03E⁻⁰⁵,PVR5B9-hIgG1—1.45E⁻⁰⁵, PVR7D4-hIgG1 5.8E⁻⁰⁵.

FIGS. 16A-16D shows the effect of the anti-PVR antibodies on tumor cellsurvival in the absence of immune cells. The survival of A549 cells(FIG. 16A), U373 cells (FIG. 16B), HCT116 cells (FIG. 16C), and Mel-624(FIG. 16D) was examined using MTT cell survival assay in the presence of50 microgram/ml of the indicated mAb for 24 hours. Significance iscalculated by a single tailed Student TTEST, *<0.05, **<0.03, and***<0.02.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monoclonal antibodies specific to thehuman poliovirus receptor (PVR). The invention also provides productionand use of the mAbs as therapeutic agents. In particular, the mAbs ofthe present invention may be used, alone or in combination with otheragents, for restoring and augmenting anti-tumor killing activity ofimmune cells, and as diagnostic reagents.

The term “antigen” as used herein refers to a molecule or a portion of amolecule capable of eliciting antibody formation and being specificallybound by an antibody. An antigen may have one or more than one epitope.The specific binding referred to above is meant to indicate that theantigen will react, in a highly selective manner, with its correspondingantibody and not with the multitude of other antibodies which may beevoked by other antigens. An antigen according to some embodiments ofthe present invention is a PVR protein.

The term “PVR” as used herein refers to the poliovirus receptor, alsoknown as CD155 (cluster of differentiation 155). The PVR is atransmembrane glycoprotein with a N-terminal signal sequence, threeextracellular immunoglobulin (Ig)-like domains, a transmembrane domainand a cytoplasmic tail. It has a molecular size of approximately 80 kDaand a structure composed of three Ig-like domains, specifically anoutermost V-like domain followed by two C2-like domains. An exemplaryPVR according to the invention is set forth in GenBank accessionnumbers: NP_001129240.1, NP_001129241.1, NP_001129242.2 and NP_006496.4.These poliovirus receptors share the sequence of the extracellulardomain and therefore can be targeted by the affinity binding moiety ofthe invention.

The term “antigenic determinant” or “epitope” as used herein refers tothe region of an antigen molecule that specifically reacts with aparticular antibody. Peptide sequences derived from an epitope can beused, alone or in conjunction with a carrier moiety, applying methodsknown in the art, to immunize animals and to produce additionalpolyclonal or monoclonal antibodies. Isolated peptides derived from anepitope may be used in diagnostic methods to detect antibodies.

It should be noted that the affinity can be quantified using knownmethods such as, Surface Plasmon Resonance (SPR) (described in ScaranoS, Mascini M, Turner A P, Minunni M. Surface plasmon resonance imagingfor affinity-based biosensors. Biosens Bioelectron. 2010, 25: 957-66),and can be calculated using, e.g., a dissociation constant, Kd, suchthat a lower Kd reflects a higher affinity.

The antibodies or a fragment thereof according to the invention binds toan epitope in hPVR. Specifically, the antibodies bind to an epitopewithin amino acids 1-343 of the PVR as set forth in NP_006496.4.

Antibodies, or immunoglobulins, comprise two heavy chains linkedtogether by disulfide bonds and two light chains, each light chain beinglinked to a respective heavy chain by disulfide bonds in a “Y” shapedconfiguration. Proteolytic digestion of an antibody yields Fv (Fragmentvariable) and Fc (Fragment crystalline) domains. The antigen bindingdomains, Fab, include regions where the polypeptide sequence varies. Theterm F(ab′)₂ represents two Fab′ arms linked together by disulfidebonds. The central axis of the antibody is termed the Fc fragment. Eachheavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains (C_(H)). Each light chain has a variabledomain (V_(L)) at one end and a constant domain (C_(L)) at its otherend, the light chain variable domain being aligned with the variabledomain of the heavy chain and the light chain constant domain beingaligned with the first constant domain of the heavy chain (CH1). Thevariable domains of each pair of light and heavy chains form theantigen-binding site. The domains on the light and heavy chains have thesame general structure and each domain comprises four framework regions,whose sequences are relatively conserved, joined by three hyper-variabledomains known as complementarity determining regions (CDRs 1-3). Thesedomains contribute specificity and affinity of the antigen-binding site.

CDR identification or determination from a given heavy or light chainvariable sequence, is typically made using one of few methods known inthe art. For example, such determination is made according to the Kabat(Wu T. T and Kabat E. A., J Exp Med, 1970; 132:211-50) and IMGT (LefrancM-P, et al., Dev Comp Immunol, 2003, 27:55-77).

When the term “CDR having a sequence”, or a similar term is used, itincludes options wherein the CDR comprises the specified sequences andalso options wherein the CDR consists of the specified sequence.

The antigen specificity of an antibody is based on the hyper variableregion (HVR), namely the unique CDR sequences of both light and heavychains that together form the antigen-binding site.

The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu)determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM,respectively). The light chain is either of two isotypes (kappa, κ orlambda, λ). Both isotopes are found in all antibody classes.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length or intact monoclonalantibodies), polyclonal antibodies, multivalent antibodies, and antibodyfragments long enough to exhibit the desired biological activity, namelybinding to human PVR.

Antibody or antibodies according to the invention include intactantibodies, such as polyclonal antibodies or monoclonal antibodies(mAbs), as well as proteolytic fragments thereof, such as the Fab orF(ab′)₂ fragments. Single chain antibodies also fall within the scope ofthe present invention.

Antibody Fragments

“Antibody fragments” comprise only a portion of an intact antibody,generally including an antigen binding site of the intact antibody andthus retaining the ability to bind antigen. Examples of antibodyfragments encompassed by the present definition include: (i) the Fabfragment, having VL, CL, VH and CH1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the CH1 domain; (iii) the Fd fragment having VH and CH1domains; (iv) the Fd′ fragment having VH and CH1 domains and one or morecysteine residues at the C-terminus of the CH1 domain; (v) the Fvfragment having the VL and VH domains of a single arm of an antibody;(vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) whichconsists of a VH domain; (vii) isolated CDR regions; (viii) F(ab′)₂fragments, a bivalent fragment including two Fab′ fragments linked by adisulphide bridge at the hinge region; (ix) single chain antibodymolecules (e.g. single chain Fv; scFv) (Bird et al., Science 1988, 242,423-426; and Huston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85,5879-5883); (x) “diabodies” with two antigen binding sites, comprising aheavy chain variable domain (VH) connected to a light chain variabledomain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90,6444-6448); (xi) “linear antibodies” comprising a pair of tandem Fdsegments (VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from antibody phage libraries. Alternatively,Fab′-SH fragments can be directly recovered from E. coli and chemicallycoupled to form F(ab′)₂ fragments (Carter et al., Bio/Technology10:163-167 (1992)). According to another approach, F(ab′)₂ fragments canbe isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner. In other embodiments, the antibody of choiceis a single chain Fv fragment (scFv).

Single chain antibodies can be single chain composite polypeptideshaving antigen binding capabilities and comprising amino acid sequenceshomologous or analogous to the variable regions of an immunoglobulinlight and heavy chain i.e. linked V_(H)-V_(L) or single chain Fv (scFv).Techniques for the production of single-chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single-chain antibodies to PVR.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigen. Furthermore, in contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” is not to be construed as requiringproduction of the antibody by any particular method. mAbs may beobtained by methods known to those skilled in the art. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohleret al., Nature 1975, 256, 495, or may be made by recombinant DNA methods(see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also beisolated from phage antibody libraries using the techniques described,for example, in Clackson et al., Nature 1991, 352, 624-628 or Marks etal., J. Mol. Biol., 1991, 222:581-597.

The design and development of recombinant monovalent antigen-bindingmolecules derived from monoclonal antibodies through rapididentification and cloning of the functional variable heavy (VH) andvariable light (VL) genes and the design and cloning of a synthetic DNAsequence optimized for expression in recombinant bacteria are describedin Fields et at. 2013, 8(6):1125-48.

The mAbs of the present invention may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, and IgD. A hybridoma producing a mAb maybe cultivated in-vitro or in-vivo. High titers of mAbs can be obtainedby in-vivo production where cells from the individual hybridomas areinjected intra-peritoneally into pristine-primed Balb/c mice to produceascites fluid containing high concentrations of the desired mAbs. mAbsmay be purified from such ascites fluids, or from culture supernatants,using methods well known to those of skill in the art.

Anti-idiotype antibodies specifically immunoreactive with thehypervariable regions of an antibody of the invention are alsocomprehended.

The invention provides a monoclonal antibody or an antibody fragmentcomprising an antigen binding domain (ABD) which comprises three CDRs ofa light chain and three CDRs of a heavy chain, wherein said ABD has atleast 90% sequence identity or similarity with an ABD of a monoclonalmouse antibody comprising: (i) a heavy variable chain comprising theamino acid sequence SEQ ID NO: 69 and a light variable chain comprisingthe amino acid sequence SEQ ID NO: 71 (herein identified as 4E5 orhPVR.07); (ii) a heavy variable chain comprising the amino acid sequenceSEQ ID NO: 73 and a light variable chain comprising the amino acidsequence SEQ ID NO: 75 (herein identified as 7D4 or hPVR.01); or (iii) aheavy variable chain comprising the amino acid sequence SEQ ID NO: 77and a light variable chain comprising the amino acid sequence SEQ ID NO:79 (herein identified as 5B9 or hPVR.09). Such antibody may have an ABDdomain having at least 93%, at least 94%, at least 95%, at least 96, atleast 97, at least 98, at least 99% sequence identity or similarity or100% sequence identity with corresponding ABD of 4E5, 7D4 or 5B9.

Sequence identity is the amount of amino acids or nucleotides whichmatch exactly between two different sequences. Sequence similaritypermits conservative substitution of amino acids to be determined asidentical amino acids.

The invention also provides conservative amino acid variants of theantibody molecules according to the invention. Variants according to theinvention also may be made that conserve the overall molecular structureof the encoded proteins. Given the properties of the individual aminoacids comprising the disclosed protein products, some rationalsubstitutions will be recognized by the skilled worker. Amino acidsubstitutions, i.e., “conservative substitutions,” may be made, forinstance, on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. The term “antibody analog” as used herein refers toan antibody derived from another antibody by one or more conservativeamino acid substitutions.

The term “antibody variant” as used herein refers to any moleculecomprising the antibody of the present invention. For example, fusionproteins in which the antibody or an antigen-binding-fragment thereof islinked to another chemical entity is considered an antibody variant.

Analogs and variants of the antibody sequences are also within the scopeof the present application. These include, but are not limited to,conservative and non-conservative substitution, insertion and deletionof amino acids within the sequence. Such modification and the resultantantibody analog or variant are within the scope of the present inventionas long as they confer, or even improve the binding of the antibody tothe human PVR.

Conservative substitutions of amino acids as known to those skilled inthe art are within the scope of the present invention. Conservativeamino acid substitutions include replacement of one amino acid withanother having the same type of functional group or side chain, e.g.,aliphatic, aromatic, positively charged, negatively charged. Thesesubstitutions may enhance oral bioavailability, penetration, andtargeting to specific cell populations, immunogenicity, and the like.One of skill will recognize that individual substitutions, deletions oradditions to a peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.For example, according to one table known in the art, the following sixgroups each contain amino acids that are conservative substitutions forone another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

It should be emphasized that the variant chain sequences are determinedby sequencing methods using specific primers. Different sequencingmethods employed on the same sequence may result in slightly differentsequences due to technical issues and different primers, particularly inthe sequence terminals. Therefore, different variants of the anti-PVRvariable chain sequences are specified along the application.

The terms “molecule having the antigen-binding portion of an antibody”and “antigen-binding-fragments” as used herein are intended to includenot only intact immunoglobulin molecules of any isotype and generated byany animal cell line or microorganism, but also the antigen-bindingreactive fraction thereof, including, but not limited to, the Fabfragment, the Fab′ fragment, the F(ab′)₂ fragment, the variable portionof the heavy and/or light chains thereof, Fab mini-antibodies (see e.g.,WO 93/15210, U.S. patent application Ser. No. 08/256,790, WO 96/13583,U.S. patent application Ser. No. 08/817,788, WO 96/37621, U.S. patentapplication Ser. No. 08/999,554), and single-chain antibodiesincorporating such reactive fraction, as well as any other type ofmolecule in which such antibody reactive fraction has been physicallyinserted. Such molecules may be provided by any known technique,including, but not limited to, enzymatic cleavage, peptide synthesis orrecombinant techniques.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species, or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In addition,complementarity determining region (CDR) grafting may be performed toalter certain properties of the antibody molecule including affinity orspecificity. A non-limiting example of CDR grafting is disclosed in U.S.Pat. No. 5,225,539.

Chimeric antibodies are molecules of which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Antibodies that have variable region framework residuessubstantially from human antibody (termed an acceptor antibody) and CDRssubstantially from a mouse antibody (termed a donor antibody) are alsoreferred to as humanized antibodies. Chimeric antibodies are primarilyused to reduce immunogenicity in application and to increase yields inproduction, for example, where murine mAbs have higher yields fromhybridomas but higher immunogenicity in humans, such that human/murinechimeric mAbs are used. Chimeric antibodies and methods for theirproduction are known in the art (for example PCT patent applications WO86/01533, WO 97/02671, WO 90/07861, WO 92/22653 and U.S. Pat. Nos.5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).

According to some specific embodiments, the monoclonal antibody is achimeric monoclonal antibody.

According to some embodiments, the chimeric antibody compriseshuman-derived constant regions.

According to some embodiments the human constant regions of the chimericantibody are selected from the group consisting of: human IgG1, humanIgG2, human IgG3, and human IgG4.

According to a specific embodiment the chimeric monoclonal antibody orfragment thereof, comprises a constant region subclass of human IgG1subtype.

According to a particular embodiment, a chimeric monoclonal antibodywhich recognizes PVR is provided comprising a set of six CDRs selectedfrom the group consisting of: (i) SEQ ID NOs: 4 or 80, 6 or 81, 8 or 82,12, 14, and, 16; (ii) SEQ ID Nos: 20 or 83, 22, 24, 28, 30, and 32; and(iii) SEQ ID Nos: 36 or 84, 38, 40, 44 or 85, 46, and 48.

Pharmacology

In pharmaceutical and medicament formulations, the active agent ispreferably utilized together with one or more pharmaceuticallyacceptable carrier(s) and optionally any other therapeutic ingredients.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and not undulydeleterious to the recipient thereof. The active agent is provided in anamount effective to achieve the desired pharmacological effect, asdescribed above, and in a quantity appropriate to achieve the desiredexposure.

Typically, the antibodies and fragments and conjugates thereof of thepresent invention comprising the antigen binding portion of an antibodyor comprising another polypeptide including a peptide-mimetic will besuspended in a sterile saline solution for therapeutic uses. Thepharmaceutical compositions may alternatively be formulated to controlrelease of active ingredient (molecule comprising the antigen bindingportion of an antibody) or to prolong its presence in a patient'ssystem. Numerous suitable drug delivery systems are known and include,e.g., implantable drug release systems, hydrogels,hydroxymethylcellulose, microcapsules, liposomes, microemulsions,microspheres, and the like. Controlled release preparations can beprepared through the use of polymers to complex or adsorb the moleculeaccording to the present invention. For example, biocompatible polymersinclude matrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebaric acid. Therate of release of the molecule according to the present invention,i.e., of an antibody or antibody fragment, from such a matrix dependsupon the molecular weight of the molecule, the amount of the moleculewithin the matrix, and the size of dispersed particles.

The pharmaceutical composition of this invention may be administered byany suitable means, such as orally, topically, intranasally,subcutaneously, intramuscularly, intravenously, intra-arterially,intraarticulary, intralesionally, intratumorally or parenterally.Ordinarily, intravenous (i.v.) administration is used for deliveringantibodies.

It will be apparent to those of ordinary skill in the art that thetherapeutically effective amount of the molecule according to thepresent invention will depend, inter alia upon the administrationschedule, the unit dose of molecule administered, whether the moleculeis administered in combination with other therapeutic agents, the immunestatus and health of the patient, the therapeutic activity of themolecule administered, its persistence in the blood circulation, and thejudgment of the treating physician.

As used herein the term “therapeutically effective amount” refers to anamount of a drug effective to treat a disease or disorder in a mammal.In the case of cancer, the therapeutically effective amount of the drugmay reduce the number of cancer cells; reduce the tumor size; inhibit(i.e., slow to some extent and preferably stop) cancer cell infiltrationinto peripheral organs; inhibit (i.e., slow to some extent andpreferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the disorder. To the extent the drug may prevent growthand/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. For cancer therapy, efficacy in vivo can, for example, bemeasured by assessing the duration of survival, time to diseaseprogression (TTP), the response rates (RR), duration of response, and/orquality of life.

The cancer amendable for treatment by the present invention includes,but is not limited to: carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high-grade immunoblastic NHL; high-gradelymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.Preferably, the cancer is selected from the group consisting of breastcancer, colorectal cancer, rectal cancer, non-small cell lung cancer,non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, livercancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma,carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer,mesothelioma, and multiple myeloma. The cancerous conditions amendablefor treatment of the invention include metastatic cancers.

According to other embodiments, the pharmaceutical composition accordingto the invention are for use in treating cancer characterized by PVRoverexpression. PVR overexpression related cancer types can beidentified using known data bases such as The Cancer Genome Atlas(TCGA). According to certain embodiments, the cancer is selected fromthe group consisting of adrenocortical carcinoma (ACC), chromophoberenal cell carcinoma (KICH), liver hepatocellular carcinoma (LIHC),colon and rectal adenocarcinoma (COAD, READ), pancreatic ductaladenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG),papillary kidney carcinoma (KIRP), lung adenocarcinoma (LUAD), head andneck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD),uterine corpus endometrial carcinoma (UCEC), cervical cancer (CESC),cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladdercancer (BLCA), clear cell kidney carcinoma (KIRC), lung squamous cellcarcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarianserous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA),glioblastoma multiforme (GBM), breast cancer (BRCA), lower grade glioma(LGG), and diffuse large B-cell lymphoma (DLBC). Each possibilityrepresents a separate embodiment of the invention.

The molecules of the present invention as active ingredients aredissolved, dispersed or admixed in an excipient that is pharmaceuticallyacceptable and compatible with the active ingredient as is well known.Suitable excipients are, for example, water, saline, phosphate bufferedsaline (PBS), dextrose, glycerol, ethanol, or the like and combinationsthereof. Other suitable carriers are well known to those skilled in theart. In addition, if desired, the composition can contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents.

The pharmaceutical composition according to the present invention may beadministered together with an anti-neoplastic composition.

The term “treatment” as used herein refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include melanoma, lung, thyroid,breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic,leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, orendometrial cancer.

According to some embodiments, the method of treating cancer comprisesadministering the pharmaceutical composition as part of a treatmentregimen comprising administration of at least one additional anti-canceragent.

According to some embodiments, the anti-cancer agent is selected fromthe group consisting of an antimetabolite, a mitotic inhibitor, ataxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, anasparaginase, an alkylating agent, an antitumor antibiotic, andcombinations thereof. Each possibility represents a separate embodimentof the invention.

According to some embodiments, the antimetabolite is selected from thegroup consisting of cytarabine, gludarabine, fluorouracil,mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea.According to some embodiments, the mitotic inhibitor is selected fromthe group consisting of vincristine, vinblastine, and vinorelbine.According to some embodiments, the topoisomerase inhibitor is selectedfrom the group consisting of topotecan and irenotecan. According to someembodiments, the alkylating agent is selected from the group consistingof busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide,cisplatin, carboplatin, ifosamide, mechlorethamine, melphalan, thiotepa,dacarbazine, and procarbazine. According to some embodiments, theantitumor antibiotic is selected from the group consisting of bleomycin,dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin,mitoxantrone, and plicamycin. According to some embodiments, thetopoisomerase II is selected from the group consisting of etoposide andteniposide. Each possibility represents a separate embodiment of thepresent invention.

According to some particular embodiments, the additional anti-canceragent is selected from the group consisting of bevacizumab, carboplatin,cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride,topotecan hydrochloride, thiotepa, and combinations thereof. Eachpossibility represents a separate embodiment of the present invention.

Monoclonal antibodies according to the present invention may be used aspart of combined therapy with at least one anti-cancer agent. Accordingto some embodiments, the additional anti-cancer agent is animmuno-modulator, an activated lymphocyte cell, a kinase inhibitor or achemotherapeutic agent.

According to some embodiments, the anti-cancer agent is animmuno-modulator, whether agonist or antagonist, such as antibodyagainst an immune checkpoint molecule.

Checkpoint immunotherapy blockade has proven to be an exciting new venueof cancer treatment. Immune checkpoint pathways consist of a range ofco-stimulatory and inhibitory molecules which work in concert in orderto maintain self-tolerance and protect tissues from damage by the immunesystem under physiological conditions. Tumors take advantage of certaincheckpoint pathways in order to evade the immune system. Therefore, theinhibition of such pathways has emerged as a promising anti-cancertreatment strategy.

The anti-cytotoxic T lymphocyte 4 (CTLA-4) antibody ipilimumab (approvedin 2011) was the first immunotherapeutic agent that showed a benefit forthe treatment of cancer patients. The antibody interferes withinhibitory signals during antigen presentation to T cells.Anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in2014) blocks negative immune regulatory signaling of the PD-1 receptorexpressed by T cells. An additional anti-PD-1 agent was filed forregulatory approval in 2014 for the treatment of non-small cell lungcancer (NSCLC). Active research is currently exploring many other immunecheckpoints, among them: CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R,lymphocyte activation gene 3 (LAG3), CD137, OX40 (also referred to asCD134), and killer cell immunoglobulin-like receptors (KIR).

According to some specific embodiments, the immuno-modulator is selectedfrom the group consisting of: an antibody inhibiting CTLA-4, ananti-human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2antibody, an activated cytotoxic lymphocyte cell, a lymphocyteactivating agent, an antibody against CEACAM, an antibody against TIGIT,and a RAF/MEK pathway inhibitor. Each possibility represents a separateembodiment of the present invention. According to some specificembodiments, the additional immuno-modulator is selected from mAb toPD-1, mAb to PD-L1, mAb to PD-L2, mAb to CEACAM1, mAb to CTLA-4, mAB toTIGIT, Interleukin 2 (IL-2) or lymphokine-activated killer (LAK) cell.

According to other embodiments the additional anti-cancer agent is achemotherapeutic agent. The chemotherapy agent, which could beadministered together with the antibody according to the presentinvention, or separately, may comprise any such agent known in the artexhibiting anticancer activity, including but not limited to:mitoxantrone, topoisomerase inhibitors, spindle poison vincas:vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel;alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide,melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil,cytarabine, gemcitabin; podophyllotoxins: etoposide, irinotecan,topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin,mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin,idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin;interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide,and megestrol acetate.

According to some embodiments, the chemotherapeutic agent is selectedfrom alkylating agents, antimetabolites, folic acid analogs, pyrimidineanalogs, purine analogs and related inhibitors, vinca alkaloids,epipodophyllotoxins, antibiotics, L-asparaginase, topoisomeraseinhibitor, interferons, platinum coordination complexes, anthracenedionesubstituted urea, methyl hydrazine derivatives, adrenocorticalsuppressant, adrenocorticosteroides, progestins, estrogens,antiestrogen, androgens, antiandrogen, and gonadotropin-releasinghormone analog. According to another embodiment, the chemotherapeuticagent is selected from the group consisting of 5-fluorouracil (5-FU),leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel anddoxetaxel. One or more chemotherapeutic agents can be used.

In some embodiments, the pharmaceutical composition according to thepresent invention is for use in treating cancer or for use in enhancingthe immune response.

The term “enhancing immune response” refers to increasing theresponsiveness of the immune system and inducing or prolonging itsmemory. The pharmaceutical composition according to the presentinvention may be used to stimulate immune system upon vaccination. Thus,in one embodiment the pharmaceutical composition can be used forimproving vaccination.

In certain embodiments, the cancer is selected from lung, thyroid,breast, colon, melanoma, prostate, hepatic, bladder, renal, cervical,pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma,biliary, and endometrial cells cancer. Each possibility represents aseparate embodiment of the invention.

According to some embodiments, a pharmaceutical composition, comprisingat least one antibody or fragment thereof according to the presentinvention, and a pharmaceutical composition, comprising an additionalimmuno-modulator or a kinase inhibitor, are used in treatment of cancerby separate administration.

According to still another aspect the present invention provides amethod of treating cancer in a subject in need thereof comprisingadministering to said subject a therapeutically effective amount of amonoclonal antibody or antibody fragment according to the presentinvention.

The term “effective amount” as used herein refers to a sufficient amountof the monoclonal antibody of the antibody fragment that, whenadministered to a subject will have the intended therapeutic effect. Theeffective amount required to achieve the therapeutic end result maydepend on a number of factors including, for example, the specific typeof the tumor and the severity of the patient's condition, and whetherthe combination is further co-administered with radiation. The effectiveamount (dose) of the active agents, in the context of the presentinvention should be sufficient to affect a beneficial therapeuticresponse in the subject over time, including but not limited toinhibition of tumor growth, reduction in the rate of tumor growth,prevention of tumor and metastasis growth and enhanced survival.

Toxicity and therapeutic efficacy of the compositions described hereincan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., by determining the IC50 (theconcentration which provides 50% inhibition) and the maximal tolerateddose for a subject compound. The data obtained from these cell cultureassays and animal studies can be used in formulating a range of dosagesfor use in humans. The dosage may vary depending inter alia upon thedosage form employed, the dosing regimen chosen, the composition of theagents used for the treatment and the route of administration utilized,among other relevant factors. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. Depending on the severity andresponsiveness of the condition to be treated, dosing can also be asingle administration of a slow release composition, with course oftreatment lasting from several days to several weeks or until cure iseffected or diminution of the disease state is achieved. The amount of acomposition to be administered will, of course, be dependent on thesubject being treated, the severity of the affliction, the manner ofadministration, the judgment of the prescribing physician, and all otherrelevant factors.

The term “administering” or “administration of” a substance, a compoundor an agent to a subject can be carried out using one of a variety ofmethods known to those skilled in the art. For example, a compound or anagent can be administered enterally or parenterally. Enterally refers toadministration via the gastrointestinal tract including per os,sublingually or rectally. Parenteral administration includesadministration intravenously, intradermally, intramuscularly,intraperitoneally, subcutaneously, ocularly, sublingually, intranasally,by inhalation, intraspinally, intracerebrally, and transdermally (byabsorption, e.g., through a skin duct). A compound or agent can alsoappropriately be introduced by rechargeable or biodegradable polymericdevices or other devices, e.g., patches and pumps, or formulations,which provide for the extended, slow or controlled release of thecompound or agent. Administering can also be performed, for example,once, a plurality of times, and/or over one or more extended periods. Insome embodiments, the administration includes both directadministration, including self-administration, and indirectadministration, including the act of prescribing a drug. For example, asused herein, a physician who instructs a patient to self-administer adrug, or to have the drug administered by another and/or who provides apatient with a prescription for a drug is administering the drug to thepatient.

Antibodies are generally administered in the range of about 0.1 to about20 mg/kg of patient weight, commonly about 0.5 to about 10 mg/kg, andoften about 1 to about 5 mg/kg. In this regard, it is preferred to useantibodies having a circulating half-life of at least 12 hours,preferably at least 4 days, more preferably up to 21 days. Chimericantibodies are expected to have circulatory half-lives of up to 14-21days. In some cases it may be advantageous to administer a large loadingdose followed by periodic (e.g., weekly) maintenance doses over thetreatment period. Antibodies can also be delivered by slow-releasedelivery systems, pumps, and other known delivery systems for continuousinfusion.

The term “about” means that an acceptable error range, e.g., up to 5% or10%, for the particular value should be assumed.

Angiogenesis

According to an aspect, the present invention provides a pharmaceuticalcomposition according to the present invention for use in treating anangiogenesis-related disease or disorder.

Angiogenesis is an important cellular event in which vascularendothelial cells proliferate, prune and reorganize to form new vesselsfrom preexisting vascular networks. There is compelling evidence thatthe development of a vascular supply is essential for normal andpathological proliferative processes and inflammation. The vascularcompartment is necessary not only for organ development anddifferentiation during embryogenesis, but also for wound healing, tissuerepair and reproductive functions in the adult.

Angiogenesis is also implicated in the pathogenesis of a variety ofdisorders, including but not limited to, tumors, proliferativeretinopathies, age-related macular degeneration, rheumatoid arthritis,and psoriasis. Angiogenesis is essential for the growth of most primarytumors and their subsequent metastasis. Tumors can absorb sufficientnutrients and oxygen by simple diffusion up to a size of 1-2 mm, atwhich point their further growth requires the elaboration of a vascularsupply. This process is thought to involve recruitment of theneighboring host mature vasculature to begin sprouting new blood vesselcapillaries, which grow toward, and subsequently infiltrate, the tumormass. In addition, tumor angiogenesis involves the recruitment ofcirculating endothelial precursor cells from the bone marrow to promoteneovascularization.

Diagnosis

The present invention further discloses methods for diagnosing andprognosing cancer.

According to an aspect, the present invention provides a diagnosticand/or prognostic method of cancer or infectious disease in a subject,the method comprises the step of determining the expression level of PVRin a biological sample of said subject using at least one antibody asdescribed herein.

The term “biological sample” encompasses a variety of sample typesobtained from an organism that may be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen, or tissuecultures or cells derived there from and the progeny thereof.Additionally, the term may encompass circulating tumor or other cells.The term specifically encompasses a clinical sample, and furtherincludes cells in cell culture, cell supernatants, cell lysates, serum,plasma, urine, amniotic fluid, biological fluids including aqueoushumour and vitreous for eyes samples, and tissue samples. The term alsoencompasses samples that have been manipulated in any way afterprocurement, such as treatment with reagents, solubilisation, orenrichment for certain components.

Determining the expression level of PVR can be performed by a labeledanti-PVR antibody as described herein. Determining the expression can beperformed, for example, by ELISA.

The method of the invention can further comprise the step of comparingsaid level of expression to a control level.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construedas limiting the scope of the invention.

EXAMPLES Experimental Procedures

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques are wellknown in the art. Other general references referring to well-knownprocedures are provided throughout this document for the convenience ofthe reader.

Example 1. High PVR Expression Correlates with Poor Prognosis

PVR and Nectin-2 are ligands for the inhibitory receptor TIGIT (FIG. 2). The results illustrate that high PVR expression levels correlatedwith poor cancer prognosis of lung cancer, breast cancer and liposarcoma(FIGS. 1A-C, respectively). GEO expression of PVR was correlated tosurvival using bioprofiling.de the relevant data sets are ID: GSE31210,GSE25055, GSE30929. Furthermore, using the same analysis, Nectin-2expression was mostly a positive marker for survival.

Example 2. Generation and Purification of Anti-PVR mAbs

In order to generate anti-PVR antibodies, a recombinant protein wasproduced and purified, hPVR-Fc, that combines extracellular part ofhuman PVR and human Fc region of an immunoglobulin G carrier as animmunogen.

BALB/c mice were injected with 50 μg of the immunogen in completeFreund's adjuvant and 2 weeks later in incomplete Freund's adjuvant.After 2 weeks, the sera were screened for the antibody titer. The bestresponders (the serum was monitored by ELISA assay for the titer of theanti-hPVR-Fc antibodies) were boosted with the immunogen in PBS. Threedays later, spleen cells were collected, and after lysis of red bloodcells, fused with SP2/0 cells. The cells were seeded in 20% RPMI 1640medium containing hypoxanthine, aminopterine, and thymidine forhybridoma selection and screened for mAbs using ELISA. Stable hybridomacell lines were generated by fusing SP2/0 myeloma cells with spleencells of an immunized mouse.

Positive outcomes (cell lines secreting antibodies that recognizehPVR-Fc) were further selected to develop a product that will haveseveral differentiating characteristics: a) high yield to reduce theantibody-production costs; b) the lack of cross-reactivity between mouseand human PVR and several other ligands of the immune cell receptors; c)a strong binding capacity to the native, mature human PVR moleculesexpressed on the surface of live cells (the antibodies were chosen froma comprehensive anti-hPVR monoclonal antibody pool with provencapability to recognize hPVR in different techniques e.g. flowcytometry, western blot, ELISA). Indeed, the human and mouse PVR havehigh level of homology and it is not easy to generate a mouse monoclonalantibody that recognizes a human homologue. More importantly, human PVRis extensively glycosylated on its extracellular region. For thesereasons, it is not easy to generate an antibody that recognizes a nativeprotein using common antigens (such as E. coli derived ones).

The present inventors have used the hPVR-Fc immunogen, a moleculeproduced in mammalian human embryonic kidney 293 (HEK 293T) cells andpurified under native conditions by immunoaffinity chromatography, toclosely mimic the native protein, human PVR. In conclusion, from a poolof generated anti-hPVR mAbs, representatives that recognize a nativehuman PVR form on live cells were identified, which is a prerequisite todevelop a derivative that would influence the human immune cell responseduring the treatment.

Four anti-PVR antibodies were generated. Of these, three antibodies wereblocking anti-PVR mAbs, namely antibody 5B9 (also termed hPVR.09),antibody 7D4 (also termed hPVR.01) and antibody 4E5 (also termedhPVR.07). These antibodies all block TIGIT-Ig binding (as illustrated inFIGS. 3B-D, respectively). A fourth antibody was generated which doesnot block TIGIT-Ig binding and was termed 2G3 (also termed hPVR.17)(FIG. 3A).

Surface plasmon resonance (SPR) Biosensor Biacore™ T100 (GE Healthcare)was used to determine Koff, Kon and K_(D) between the antibodies andhPVR (Table 1).

TABLE 1 Antibodies affinity measurement by Biacore ™ mAB Affinity 4E57.22E⁻¹⁰ 5B9 1.62E⁻⁰⁹ 7D4 1.93E⁻¹⁰

Chimeric monoclonal antibodies, comprising human heavy chain constantIgG1 region set forth in SEQ ID NO: 86 (corresponding to GenBank:AAA02914.1), were produced from the above three antibodies, usingmethods known in the art.

Example 3. Blocking of PVR-TIGIT Interactions with Anti-PVR mAb EnhancedNK Cell Killing of Human Cell Lines

The target cells were labeled with [35S]-Methionine 12 hours prior tothe assay. Indicated antibodies were added to the final concentration of5 μg/ml and incubated with the labeled targets (5000 cells/well) for 30minutes on ice. The assays were performed in RPMI medium in 96U shapedplates at 37° C. for 5 hours. Labeled targets were incubated witheffector NK cells at 10:1 E:T ratio. Following incubation, plates werecentrifuged (1600 rpm, 5 min, 4° C.) and supernatants (50 μl) werecollected and transferred to opaque Opti-plates (Packard). 150 μlscintillation liquid (Perkin Elmer) was added and analyzed by a microbeta, β-counter (Perkin Elmer). The maximal labeling was determined byadding 100 μl of 0.1N NaOH to an equal amount of targets (5000/well).Spontaneous release was determined in wells containing target cellsonly. Final specific killing was calculated as follows: ((radioactivereading−spontaneous release)/(maximal labeling−spontaneousrelease))*100=specific killing. As shown in FIG. 4 , culturing of NKcells with the anti-PVR mAb 7D4 (also termed hPVR.01) enhanced (by twofolds) NK cell killing of the human breast Adenocarcinoma cell lineMDA-MB-231.

Example 4. Blocking of PVR-TIGIT Interactions with Anti-PVR mAb EnhancedNK Cell Killing of Human Cancer Cell Lines

Target cells were labeled with [35S]-Methionine 12 hours prior to theassay. Indicated antibodies were added to the final concentration of 5μg/ml and incubated with the labeled targets (5000 cells/well for 30minutes on ice. The cells were incubated with effector NK cells at 10:1E:T ratio. The assays were performed in RPMI medium in 96-U shapedplates at 37° C. for 5 hours. Following incubation, plates werecentrifuged (1600 rpm, 5 min, 4° C.) and supernatants (50 μl) werecollected and transferred to opaque Opti-plates (Packard). 150 μlscintillation liquid (Perkin Elmer) was added and analyzed by a microbeta, 0-counter (Perkin Elmer). The maximal labeling was determined byadding 100 μl of 0.1N NaOH to an equal amount of targets (5000/well).Spontaneous release was determined in wells containing target cellsonly. Final specific killing was calculated as follows: ((radioactivereading−spontaneous release)/(maximal labeling−spontaneousrelease))*100=specific killing.

As shown in FIG. 5 , blocking of PVR-TIGIT interactions with theanti-PVR mAb 4E5 (also termed hPVR.07) enhances NK cell killing of humancancer cell line HepG2 (hepatocellular carcinoma). It is thus clear thatblocking of PVR leads to enhanced killing of the target cells. Thekilling is further enhanced when the human IgG counterpart of 4E5 wasused in a chimeric version of the mAb. The killing is equivalent to thepositive control (Erbitux©).

Example 5. Human Tumor Cell Lines Express PVR and Nectin-2

To examine the expression of PVR and Nectin-2 on tumor cells, expressionlevels of these proteins on different tumor cell lines was examined byFACS analysis using the anti PVR-4E5 Ab and the anti-Nectin-2 Ab (cloneTX-31), both at 2 μg/ml. As shown in FIGS. 6A-O, various human tumorcell lines express PVR and Nectin-2. Specifically, it is shown thatmelanoma cells (FIGS. 6A-E), breast cancer cells (FIGS. 6F-H),colorectal cells (FIG. 6I), kidney cells (FIG. 6J), lung cancer cells(FIG. 6K), prostate cancer cells (FIG. 6L), brain tumor cells (FIG. 6M),and hepatocellular carcinoma cells (FIGS. 6N-0 ) all express PVR andNectin-2.

Example 6. PVR is the Main TIGIT Ligand

FIGS. 7A-7C demonstrate that PVR is the main TIGIT ligand. Specifically,it was shown that HepG2 cells (human hepatocellular carcinoma cells)express both PVR and Nectin-2 (FIG. 7A). Culturing of HepG2 cells withpurified anti-PVR mAb 4E5, also termed hPVR.07 (0.15 μg/well), almostcompletely blocked TIGIT-Ig binding (2 μg/ml) (FIG. 7B), despite thefact that these cells also express Nectin-2. As shown in FIGS. 7C and7D, it is clear that there was no direct recognition of Nectin-2 byanti-PVR mAbs.

Example 7. Binding of mAb Clones to Human and Primate PVR

As shown in FIGS. 8A-C, all anti-PVR antibody clones tested bind tohuman PVR (hPVR), using FACS analysis. Briefly, cells were trypsinaizedand transferred for staining at 2*10⁵ cell per well. Indicatedantibodies were added for 30 min on ice. All antibodies were used atfinal concentration of 2 μg/ml. Bound anti-PVR Ab detection wasperformed using anti-mouse IgG-647. Mouse IgG1 kappa was used as anegative control.

FIG. 8A illustrates that endogenous hPVR was detected by FACS stainingon the surface of HepG2 hepatocellular carcinoma cells. In FIG. 8B themurine cell line B16 was used. Murine PVR sequence is different from thehuman species and is not recognized by the anti-human PVR antibodies ascan be seen by the lack of signal (left panel). When the full-lengthhPVR protein (NP_006496.4 amino acids 1-418) was overexpressed in thesecells All three clones resulted in identical signal (right panel)further supporting the claim that this staining is the result of aspecific binding of these Abs to the human PVR protein.

PVR amino acid sequence is conserved across some species. The amino acidconservation of human PVR was compared to that of African green monkeysin-silico and was found to have a similarity of 93% to human PVR. FACSstaining of African green monkey Vero cells demonstrated that themonkey's PVR is efficiently recognized by all three human mAbs (FIG.8C). It was further found that these human anti-PVR antibodies do notrecognize PVR from canine and rodents such as hamster or mouse. FIG. 9shows FACS analysis for the canine PVR expressing MDCK cells. As can beseen, none of the anti-human Abs resulted in a positive signal, whilePVR expression itself is suggested by the strong TIGIT-Ig signal.

Example 8. Nectin-2 is Preferentially Bound by DNAM-1

TIGIT-Fc and DNAM-1-Fc were used at the indicated concentrations in FIG.10 to stain cells overexpressing hNectin-2: RPMI-8866 cell line (FIGS.10A and 10B), (or B16-hPVR cell line (FIGS. 10C and 10D). Bound fusionprotein detection was made using anti-human-IgG APC and analyzed byFACS.

For the PVR binding the signal by TIGIT-Fc was 2-4 folds higher thanthat of the DNAM-1-Fc, similarly to a previous report (Yu. X et al.,2009, Nat Immunol., 10(1):48-57). At the same time, the binding ofhNectin-2 to DNAM-1-Fc was 2-10 folds stronger than its binding toTIGIT-Fc, which is contradictory to one report (Yu. X et al 2009), butcorroborated by another study (Zhu Y et al., 2016, J Exp Med., 8;213(2):167-76). Taken together, the results illustrate that DNAM-1 isthe preferential receptor for Nectin-2 binding. Accordingly, blocking ofPVR will prevent the inhibitory signaling of TIGIT, while allowing theco-stimulatory signaling by DNAM-1. DNAM-1 mediates cellular adhesion toother cells bearing its ligands.

Example 9. Anti PVR Antibodies Enhance T Cell Proliferation

To test the effect of the anti PVR mAbs on T cell proliferation, humanperipheral blood mononuclear cell (PBMC) were stained withcarboxyfluorescein succinimidyl ester (CFSE) and incubated with targetcells in the present of antibodies at a concentration of 4 μg/ml. CFSEdilution was measured on CD45 positive cells after 5-9 days in culture.As shown in FIG. 11 , Anti PVR 5B9 activity exceeds the PD-1 and CTLA4antibodies activity as a single agent. Also, chimeric anti PVR 4E5hIgG1clone, having a human IgG1 constant region, was superior to its mousecounterpart. Next, the combined effect of anti PVR mAbs and otherantibodies that were found to enhance T cell proliferation was examined.Human PBMC were stained with Carboxyfluorescein succinimidyl ester(CFSE) and incubated with target cells in the present of antibodies at aconcentration of 4 μg/ml. CFSE dilution was measured on CD45 positivecells after 5-9 days in culture. The results show that the proliferationactivity of anti-PVR 5B9, when combined with either PD-1 or CTLA-4,exceeds the activity of a combination of PD-1 and CTLA4 (FIG. 12 ).Also, the activity of a combination of anti-PVR 4E5 and CTLA-4 equalsPD-1 and CTLA4 combination. Next, the specific induction of CD8 wasexamined. As shown in FIG. 13 , Anti PVR antibodies CD8 T cellproliferation activity exceeds the activity of PD1. Also, the inductionactivity of anti PVR 5B9 antibody exceeds that of CTLA1. The ratio ofCD8/CD4 proliferation was evaluated for the different antibodies (FIG.14 ). Anti PVR 5B9 had the highest CD8/CD4 ratio. The DNA sequence ofthe variable heavy and light chains were used to construct chimericantibodies, comprising the human IgG1 isotype constant domains andconstant light (CL) human IgG Kappa domain. T-cell proliferation inducedby mouse and human chimeric counterpart antibodies was measured by theCFSE assay. The numbers shown in Table 2 represent relative level ofproliferation compared to control.

TABLE 2 A summary of the effect of anti PVR antibodies on T cellproliferation. Name of Effect on T cell Effect on T cell clone Fc Typeproliferation (CFSE) proliferation cell count 4E5 Mouse IgG1 198% 320%Human IgG1 275% 353% 5B9 Mouse IgG1 300% 410% Human IgG1 270% 364% 7D4Mouse IgG1 141% 260% Human IgG1 280% 360%

Next, the PVR-antibodies effect on NK degranulation was examined. Duringdegranulation, cytolytic granules in NK cells are released and thelysosome-associated membrane protein-1 (LAMP-1, CD107a) which is presenton cytolytic granules surface is transported to the cell surface andbecomes accessible for antibody binding. This marker allowsidentification of activated NK cells. NK cells were incubated with 5different target cells, together with the anti PVR antibodies (mouse andtheir chimeric human counterpart antibodies). Degranulation wasevaluated using anti-CD107a antibodies.

TABLE 3 Effect of anti-PVR antibodies on NK degranulation activity.Target cancer cells MDA- Clone Fc Type MB-231 HepG2 MV-411 Mel-624* A5494E5 Mouse IgG1 125% 150% 150% 120% — Human IgG1 305% 260% 160% 145% 260%5B9 Mouse IgG1 132% 160% 150% 120% — Human IgG1 300% 260% 220% 145% 240%7D4 Mouse IgG1 124% 160% 150% 115% — Human IgG1 220% 200% 130% 120% 165%*Expression of hPVR in Mel 624 is low therefore the relative effect islow.

As shown in Table 3 and FIG. 15 , all antibodies showed an NKdegranulation activity, wherein the chimeric antibodies had asignificantly higher activity compared to their corresponded mouseantibody.

Example 10. Anti PVR Antibodies Reduce the Survival of Tumor Cells inthe Absence of Immune Cells

The survival of A549, U373, HCT116, and Mel-624 cells was examined usingMTT cell survival assay in the presence of 50 microgram/ml of differentanti-PVR mAb for 24 hours. As shown in FIGS. 16A-16D and Table 4, PVRblocking by 5B9 mAb significantly reduced viability of 20-40% comparedto mIgG.

TABLE 4 Effect of anti-PVR antibodies on survival of tumor cells.Percentage of dead cells within 24 hrs across several target cell linesrelative to mIgG treated cells. mAb MDA-MB-231 HCT-116 Mel-624 A549 4E512% 20% — 25% 5B9 20% 22% 32% 25% 7D4 17% 27% 12% 25%

Example 11. In-Vivo Effect of Anti-PVR Antibodies on Human Tumor in aHumanized Mouse Model—Short Term Humanization

The anti-tumor efficacy of the antibodies is studied in vivo. Toestimate the efficacy of the antibodies described herein in inhibitionof human cancer, the antibody is studied in a model combining bothtumors and lymphocytes of human origin. NOD scid gamma (NSG) mice areengrafted with hPBMCs to restore immune-competence and challenged withhuman cancer cells. At predetermined time point/tumor size mice aretreated with anti human PVR antibody according to the invention,administered in multiple doses at different time-points post tumorchallenge. Same experiments are performed with chimeric anti-PVRantibodies. Tumor growth curves and body weight are measured 3×/week andupon sacrificing the mice, extensive phenotypic analysis of TILs andimmune populations in different organs is performed.

A similar model with tumor lines in PBMC huNSG mice is performedaccording to Gupta P., Oncoimmunology. 2015 Mar. 6; 4(2): e981449.

Example 12. In-Vivo Effect of Anti-PVR Antibodies on Human Tumor in aHumanized Mouse Model—Long Term Humanization

To estimate the efficacy of anti-PVR antibodies in inhibition of humancancer, the antibody is studied in a model combining both tumors andlymphocytes of human origin. Newborn NOD scid gamma (NSG) pups areirradiated and engrafted with CD34+ HSC to restore immune-competence.1-2 weeks after determination of immune cells reconstitution, mice arechallenged with human cancer cells and at predetermined time point/tumorsize treated anti human PVR antibody according to the invention,administered in multiple doses on different time points. Sameexperiments are performed with humanized anti-PVR antibodies. Tumorgrowth curves and body weight are measured 3×/week. Upon sacrificing themice, extensive phenotypic analysis of TILs and immune populations indifferent organs is performed.

A similar model used to study the tumor inhibitory activity of theantibodies of the present invention, was established by The JacksonLaboratory(http://immune-checkpoint.com/wp-content/uploads/sites/24/2015/01/Day-1-15.45-Rick-Huntress.pdf).

Example 13. Induction of IFNγ Secretion

To test the effect of the anti PVR mAbs on cytokine secretion, humanperipheral blood mononuclear cell (PBMC) from 2 healthy donors wereincubated with target cells in the present of antibodies (mIgG, 5B9mIgG,anti CTL-4 antibody termed Ipilimumab), at concentrations of 1 and 0.1μg/ml. Levels of IFNγ after 6 days in culture were measured. Significantinduction of IFNγ by the anti-PVR 5B9 antibody was observed(P=7.34548E⁻¹¹ for 1 μg/ml and 2.73179E⁻⁰⁸ for 0.1 μg/ml).

The secretion of IFNγ is a key component of anti-tumor immunity,induction of IFNγ secretion by anti-PVR mAbs indicates potentialadditional anti-tumorigenic effect of these compounds in cancertreatment.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation.

1. A method of delivering an isolated monoclonal antibody or an antibodyfragment thereof to a cell comprising contacting the cell with theisolated monoclonal antibody or antibody fragment thereof, wherein theisolated monoclonal antibody or antibody fragment thereof comprises aCDR set, the CDR set comprising a heavy chain (HC) CDR1, a heavy chain(HC) CDR2, a heavy chain (HC) CDR3, a light chain (LC) CDR1, a lightchain (LC) CDR2, and a light chain (LC) CDR3 selected from the groupconsisting of: i. the HC CDR1 sequence comprises a sequence selectedfrom the group consisting of GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQID NO: 84); the HC CDR2 sequence comprises EIFPGSGRINFNEKFKG (SEQ ID NO:38); the HC CDR3 sequence comprises TKIYGNSFDY (SEQ ID NO: 40); the LCCDR1 comprises a sequence selected from the group consisting ofKASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); the LC CDR2sequence comprises a sequence selected from the group consisting of:WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID NO: 56), WASSRHR (SEQ ID NO:57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO: 59), WASSRHP (SEQ IDNO: 60), and WASSRHT (SEQ ID NO: 61); and the LC CDR3 sequence comprisesQQYSRYPLT (SEQ ID NO: 48); ii. the HC CDR1 sequence comprises a sequenceselected from the group consisting of GFDFSRYW (SEQ ID NO: 4) and RYWMT(SEQ ID NO: 80); the HC CDR2 sequence comprises a sequence selected fromthe group consisting of EIHPDSSKINYTPSQ (SEQ ID NO: 6) andEIHPDSSKINYTPSQKD (SEQ ID NO: 81); the HC CDR3 sequence comprises asequence selected from the group consisting of PDGNYNALDYW (SEQ ID NO:8) and PDGNYNALDY (SEQ ID NO: 82); the LC CDR1 sequence comprisesKASQDVGTAVT (SEQ ID NO: 12); LC CDR2 is WASTRHT (SEQ ID NO: 14); and theLC CDR3 sequence comprises QQYSRYPYT (SEQ ID NO: 16); and iv. the HCCDR1 sequence comprises a sequence selected from the group consisting ofGYTFTEYTMH (SEQ ID NO: 20) and EYTMH (SEQ ID NO: 83); the HC CDR2sequence comprises GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); the HC CDR3sequence comprises VIPLEY (SEQ ID NO: 24); the LC CDR1 sequencecomprises KASQNVYTNVA (SEQ ID NO: 28); the LC CDR2 sequence comprisesSASYRYR (SEQ ID NO: 30); and the LC CDR3 sequence comprises QQYNSYPLA(SEQ ID NO: 32).
 2. The method of claim 1, wherein the HC CDR1 comprisesthe sequence SNYWIE (SEQ ID NO: 84); HC CDR2 comprises a sequence setforth in EIFPGSGRINFNEKFKG (SEQ ID NO: 38); and HC CDR3 comprises thesequence: TKIYGNSFDY (SEQ ID NO: 40).
 3. The method of claim 2, whereinthe HC CDR1 sequence comprises GYTFSNYWIE (SEQ ID NO: 36); the HC CDR2sequence comprises EIFPGSGRINFNEKFKG (SEQ ID NO: 38); the HC CDR3comprises TKIYGNSFDY (SEQ ID NO: 40); the LC CDR1 sequence comprisesKASQDVGTAVV (SEQ ID NO: 44); the LC CDR2 sequence comprises WASSRHE (SEQID NO: 59); and the LC CDR3 sequence comprises QQYSRYPLT (SEQ ID NO:48).
 4. The method of claim 1, wherein the isolated monoclonal antibodyis capable of inhibiting the binding of PVR to T cell immunoreceptorwith Ig and ITIM domains (TIGIT).
 5. The method of claim 1, wherein thecells are tumor cells.
 6. The method of claim 1, comprisingadministering the isolated monoclonal antibody or antibody fragmentthereof to cells of a subject.
 7. The method of claim 6, wherein thesubject is a human subject.
 8. The method of claim 6, wherein the cellsare of a cancer overexpressing PVR.
 9. The method of claim 6, whereinthe cells are of a cancer selected from the group consisting of amelanoma, a breast cancer, an ovarian cancer, a pancreatic cancer, acolorectal cancer, a colon cancer, a cervical cancer, a kidney cancer, alung cancer, a thyroid cancer, a prostate cancer, a brain cancer, arenal cancer, a throat cancer, a laryngeal carcinoma, a bladder cancer,a hepatic cancer, a fibrosarcoma, an endometrial cells cancer, aglioblastoma, sarcoma, a myeloid, a leukemia and a lymphoma.
 10. Themethod of claim 6, further comprising administering to said subject anadditional immuno-modulator, activated lymphocyte cell, kinaseinhibitor, chemotherapeutic agent or any other anti-cancer agent. 11.The method of claim 10, wherein the additional immune-modulator is anantibody against an immune checkpoint molecule selected from the groupconsisting of PD-1, CTLA-4, PDL-1, CEACAM1, NKG2A, B7-H3, B7-H4, VISTA,CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also referredto as CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT,and any combination thereof.
 12. The method of claim 10, wherein theanti-cancer agent is selected from the group consisting of an anti PD-1antibody, anti CTLA-1 antibody and an epidermal growth factor receptor(EGFR) inhibitor.
 13. The method of claim 10, wherein the anti-canceragent is selected from the group consisting of: Erbitux, cytarabine,fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine,gemcitabine, vincristine, vinblastine, vinorelbine, carmustine,lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin,ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine,bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin,mitomycin, mitoxantrone, plicamycin, etoposide, teniposide and anycombination thereof.
 14. The method of claim 1, wherein the monoclonalantibody is a chimeric antibody.
 15. The method of claim 1, wherein themonoclonal antibody is attached to a cytotoxic moiety, a radioactivemoiety, or an identifiable moiety.
 16. An isolated monoclonal antibodyor an antibody fragment thereof comprising a heavy chain variable regionselected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 77,or an analog having at least 95% sequence similarity with said heavychain variable region sequence, and a light chain variable sequenceselected from the group consisting of SEQ ID NO: 42 and SEQ ID NO: 79,or an analog having at least 95% sequence similarity with said lightchain variable region sequence.
 17. The isolated monoclonal antibody ofclaim 16, wherein the antibody is a chimeric antibody comprising a heavychain variable region of SEQ ID NO: 77 and a light chain variable regionof SEQ ID NO:
 79. 18. A method of delivering the isolated monoclonalantibody or an antibody fragment thereof of claim 16 to a cell of asubject, said method comprising administering the isolated monoclonalantibody to the subject.